cassava starch as modified release excipient in selected
TRANSCRIPT
Cassava starch as modified release excipient in selected gliclazide oral dosage forms
WC du Preez
B.Pharm
21638098
Dissertation submitted in fulfilment of the requirements for the degree Magister Scientiae of Pharmaceutics at the
Potchefstroom Campus of the North-West University
Supervisor: Dr JM Viljoen
Co-Supervisor: Prof JH Steenekamp
November 2015
1
Our deepest fear is not that we are inadequate. Our deepest fear is that we
are powerful beyond measure. It is our light, not our darkness, that most
frightens us. We ask ourselves, who am I to be brilliant, gorgeous, talented,
fabulous? Actually, who are you not to be? You are a child of God. Your
playing small doesn't serve the world. There's nothing enlightened about
shrinking so that other people won't feel insecure around you. We are all
meant to shine, as children do. We were born to make manifest the glory of
God that is within us. It's not just in some of us; it's in everyone. And as we
let our own light shine, we unconsciously give other people permission to do
the same. As we're liberated from our own fear, our presence automatically
liberates others.
Marianne Williamson
~||~
Dedicated to those who’ve never stopped praying for me,
my mentors and loved ones.
2
FOREWORD
A great journey has many steps and obstacles. To attempt such a journey requires, patience,
perseverance, passion and fortitude. Each of these is necessary but they alone cannot sustain
any endeavor as this. Where, these personal strengths falter support, care and guidance from
loved ones and mentors, sustain you through trying times, times when failure seems to be the
inevitable outcome. This in mind, I would like to give my sincerest gratitude to the following:
My Lord and Sheppard, thank You for the guidance I have received in this journey of discovery.
I thank You, for the perseverance, tenacity and fortitude I have been blessed with in order to
attempt and complete this chapter in my life. I thank You, for my faith and the path You have
lead me since the first day I have met You.
My late grandfather, Schalk, and my grandmother, Vonnie, thank you for all your love, pride and
endless prayers.
My parents, Este and Koos, thank you for your sacrifice, support, arguments and love that has
guided me all the way, making this chapter in my life possible.
Tannie Suzette and my brother Werner, thank you for your strength, prayers and faith in me.
All my friends, named and unnamed, you all gave me life in these few years, all the coffee, all
the laughs and all the tears.
A few in particular, Lezaan-marie Erasmus, Trizel du Toit, Lonette Wallis, Carlemi Calitz, Ruan
Joubert, Elizca Pretorius, Jacques Scholtz, Zandré Smith, Gerdus Kruger, Thokozile Okaecwe,
Angelique Lewies, Jaco Wentzel and Alissa Jooste, thank you for the advice, strength and
friendship. This experience could not be attempted or survived without it or without your special
“sanity”.
All my colleagues, fellow postgraduate students and friends in the office, you have made my
days full of life, insight and wisdom. The camaraderie and friendship was vital to complete this
degree.
Dr. Joe Viljoen, my study leader and postgraduate mother, who I might have driven mad at one
point or another; constantly popping in with coffee in hand and a chat, thank you for your faith in
my abilities, the patience with my writing and the firm though tender kindness you have shown
me every day since we’ve met.
3
Prof. Jan Steenekamp, thank you for making my choice in specialty so easy, if not for your pride
in pharmaceutics, I would certainly not have pursued this field or succeeded in it. Thank you for
all the assistance with the dissolution studies, templates and financial support.
Prof. Sandra van Dyk, thank you for your support, both financially and professionally.
Dr. Louwrens Tiedt, I thank you for your facilities and help with my SEM and micrographs.
Dr. Frans Smith, from pharmaceutical chemistry, thank you for your assistance in understanding
my IR-spectra and helping me with the FTIR-analysis.
Niel Barnard and SPIN®, thank you for all the physical analysis you helped me with.
Anriette Pretorius, the librarian, thank you for all the assistance in finding hard to come by
articles.
Jacques Scholtz and the late, Jaco van der Colff, thank you for input and willingness to help me
with my beads and tablets.
Lizl du Toit and Etienne Marais, thank you for helping me understand and use the
UV-spectrometer.
The “Tannies”, Dr. Maides Malan and Mrs. Mariette Fourie, thank for all your kindness, wisdom
and guidance you have given me.
Liezl (Lee) Badenhorst, thank you for all the social events and the opportunity to be your demi.
Pharmacen® including its members, associates and facilities, for affording me the opportunity to
conduct and complete a postgraduate degree.
These are just a few words of gratitude, they cannot convey the full extent and breadth of my
feelings in regard, to each participant and individual named, unnamed, known and unknown.
4
TABLE OF CONTENTS
Foreword .................................................................................. 2
Table of Contents .................................................................... 4
1.1 Aim .................................................................................. 16
1.2 Background .................................................................... 16
1.3 Objectives ....................................................................... 19
2.1 Introduction .................................................................... 20
2.1.1 Treatment of type 2-diabetes ...................................................... 22
2.1.1.1 Gliclazide ..............................................................................................................23
2.2 Solid Oral Dosage Forms ................................................ 25
2.2.1 Formulation of solid oral dosage forms ....................................... 25
2.2.1.1 Excipients used in formulations ............................................................................26
2.2.2 Manufacturing methods of solid oral dosage forms ................... 28
2.2.2.1 Wet granulation ....................................................................................................28
2.2.2.2 Dry granulation .....................................................................................................29
2.2.2.3 Direct compression ...............................................................................................29
2.2.2.4 Extrusion-Spheronised pharmaceutical pellets .....................................................29
2.3 Immediate release compared to modified release solid
oral dosage forms .......................................................... 30
2.3.1 Types of immediate release solid oral dosage forms ................. 31
2.3.1.1 Conventional release solid oral dosage forms ......................................................31
2.3.1.2 Effervescent Tablets .............................................................................................31
2.3.1.3 Chewable Tablets .................................................................................................32
2.3.1.4 Sublingual and Buccal Tablets..............................................................................32
2.3.1.5 Multi-layer tablets .................................................................................................33
2.3.1.6 Lozenges ..............................................................................................................33
2.3.2 Modified release solid oral dosage forms ................................... 33
2.3.2.1 Coated tablets ......................................................................................................33
5
2.3.2.2 Diffusion-controlled tablets ...................................................................................34
2.3.2.3 Dissolution-controlled tablets ................................................................................34
2.3.2.4 Erosion controlled tablets .....................................................................................34
2.3.2.6 Osmosis controlled tablets ....................................................................................34
2.3.2.7 Multi-layer tablets .................................................................................................35
2.3.2.8 Multi-particulates ..................................................................................................36
2.3.2.8.1 Layering .............................................................................................................37
2.3.2.8.2 Freeze pelletisation ............................................................................................37
2.3.2.8.3 Cryopellitisation ..................................................................................................38
2.3.2.8.4 Hot-melt extrusion ..............................................................................................38
2.3.2.8.5 Extrusion-spheronisation ....................................................................................38
2.4 Starch as a versatile excipient ...................................... 39
2.4.1 Cassava ......................................................................................... 41
2.5 Summary ......................................................................... 44
3.1 INTRODUCTION .............................................................. 45
3.2 Materials ......................................................................... 45
3.3 Characterisation of cassava starches ........................... 46
3.3.1 Thermoanalytical characterisation ............................................. 47
3.3.1.1 Differential scanning calorimetric (DSC) analysis ..................................................47
3.3.1.2 Thermogravimetric analysis ..................................................................................47
3.3.1.3 Karl-Fischer titration .............................................................................................48
3.3.2 Infrared (IR) analysis .................................................................... 48
3.4 Solid oral dosage forms.................................................. 49
3.4.1 Preparation of beads .................................................................... 49
3.4.1 Morphology of powder particles and bead formulations ............ 50
3.4.1.1 Scanning electron microscopy (SEM) ...................................................................50
3.4.1.2 Particle size analysis ............................................................................................51
3.5 Flow properties .............................................................. 52
3.5.1 Critical orifice diameter ............................................................... 52
6
3.5.2 Flow rate ....................................................................................... 53
3.5.4 Angle of repose ............................................................................. 54
3.5.4 Powder density ............................................................................. 55
3.5.5 Compressibility ............................................................................. 56
3.6 Evaluation of the bead formulations .............................. 57
3.6.1 Friability ........................................................................................ 57
3.6.2 Swelling and mass loss ................................................................ 57
3.6.3 Disintegration ............................................................................... 58
3.6.4 Ultraviolet-spectrophotometric analysis ..................................... 58
3.6.4.1 Standard curve .....................................................................................................59
3.6.4.1.1 Interday precision ...............................................................................................59
3.6.4.1.2 Intraday precision ...............................................................................................59
3.6.5 Dissolution Behaviour .................................................................. 59
3.6.5.1 Assay ...................................................................................................................60
3.6.5.2 Dissolution studies ................................................................................................60
3.7 Statistical analysis ......................................................... 60
3.8 Summary ......................................................................... 61
4.1 Introduction .................................................................... 63
4.2 Physical characteristics of Cassava starch .................. 64
4.2.1 Moisture content and Thermal analysis ...................................... 64
4.2.2 Infrared-spectroscopy .................................................................. 67
4.3 preliminary experiments and bead manufacturing ....... 68
4.4 Morphology and size ....................................................... 72
4.4.1 Morphology ................................................................................... 72
4.4.2 Size distribution of powder particles .......................................... 75
4.5 Flow properties .............................................................. 76
7
4.6 Evaluation of bead formulations .................................... 78
4.6.1 Friability ........................................................................................ 79
4.6.2 Swelling and mass loss ................................................................ 80
4.6.3 Disintegration ............................................................................... 82
4.6.4 Dissolution behaviour and statistical analyses .......................... 82
4.6.4.1 Standard curve .....................................................................................................82
4.6.4.2 Linearity ................................................................................................................82
4.6.4.3 Dissolution ............................................................................................................83
4.7 Summary ......................................................................... 86
5.1 SUMMARY & FUTURE PROSPECTS ............................... 88
5.2 FUTURE PROSPECTS ..................................................... 90
References............................................................................. 91
Annexure A .......................................................................... 108
Annexure B .......................................................................... 116
Annexure C .......................................................................... 128
8
LIST OF FIGURES
Figure 2.1: Chemical structure of gliclazide
Figure 2.2: Arbitrary graph comparing immediate and controlled drug release
Figure 2.3: Sublingual and buccal route of administration
Figure 2.4: Example of an osmotically controlled release tablet
Figure 2.5: Layering process for pharmaceutical beads
Figure 2.6: Radial extruder
Figure 2.7: Molecular and macroscopic structure of amylose and amylopectin
Figure 2.8: Illustration of cassava plant and root
Figure 3.1: Apparatus used for the critical orifice diameter determination
Figure 3.2: Angle of repose of a resting powder heap
Figure 4.1: Average moisture content of donated and the purchased Cassava starch, at
40°C for various drying times
Figure 4.2: Thermograms of donated Cassava starch
Figure 4.3: Thermograms of purchased Cassava starch
Figure 4.4: Overlay of IR-spectra for the donated and purchased starch
Figure 4.5: IR-spectra form FTIR of the donated and purchased Cassava starch
Figure 4.6: Scanning electron micrographs of purchased and donated starch
Figure 4.7: SEM-micrographs of the different bead formulations
Figure 4.8: Size distribution histograms of starches and beads
Figure 4.9: Cumulative mass increase or decrease of Avicel® beads as a function of time
(min) after exposure to calibrated pH environments
Figure 4.10: Standard curve of gliclazide dissolved in 2:3 methanol:HCl solution
Figure 4.11: Percentage of the drug dissolved as a function of time (min) within pH calibrated
medium form simulating either a acidic or alkaline gastric environments
9
Figure A.I: Example graphs of size distribution graphs for starch powders and bead
formulation
10
LIST OF TABLES
Table 2.1: Physicochemical properties of gliclazide
Table 2.2: Excipient types and examples
Table 2.3: Content properties of cassava
Table 2.4: Physicochemical properties of cassava variants
Table 3.1: Pharmaceutical materials employed in the various formultaions, batch numbers
and suppliers
Table 3.2: Variables and different levels of each variable as employed in this study.
Table 3.3: Flow quality of powders for various angles of repose
Table 3.4: Flow quality as indicated by Carr’s index and the Hausner ratio
Table 4.1: Identifiers for each bead formulation and the composition of each formulation
Table 4.2: Selected formulations and respective excipients and concentration
Table 4.2: Flow properties of starches and bead formulations
Table 4.3: Percentage friability of bead formulations
Table 4.4: Mean dissolution time and similarity factor values for each bead formulation and
Diamicron®
Table A.I: Karl-Fischer titration values for moisture content of the donated Cassava starch
Table A.II: Karl-Fischer titration values for moisture content of the purchased Cassava
starch
Table A.III: Size distribution values of starch powders and bead formulations
Table B.I: Time and flow rate for Cassava starch powders and bead formulations
Table B.II: Parameters and values relating to angle of repose, the angle of repose and
critical orifice diameter
Table B.III: Volumes, densities and compressibility data
Table B.IV: Swelling and erosion data
11
Table B.V: Friability parameters and data
Table B.VI: Disintegration times
Table C.I: Linearity and validation data for gliclazide in acidic medium
Table C.II: Linearity and validation data for gliclazide in alkaline medium
Table C.III: Dissolution study data for bead formulations and Diamicron®
12
Abstract
Cassava starch as modified release excipient in oral dosage
forms using gliclazide as model drug
Solid oral dosage forms are still the most leading delivery system employed commercially due to
the ease in which it can be handled, administered and even transported. Several varieties of
solid oral dosage forms are commercially available which include different types of tablets,
capsules, multi-unit particulate systems as well as medicated lozenges. Different designs and
manufacturing methods are used for solid oral dosage forms resulting in different release
mechanisms. Drug release is an important consideration during dosage form design especially
for drugs with short half-lives. These types of drugs require regularly timed dosing intervals.
More dose intervals can impede the adherence to therapy, because patients might forget a
dose. The lack in adherence adversely affects the treatment protocol necessary for the
management of disease. To overcome adversities and to modify drug release, various methods
can be employed in order to provide a desirable therapeutic product, including alternative
manufacturing methods and the addition of specialised excipients. One of the most promising
manufacturing methods to date regarding modified release, whether sustained, controlled or
multi-dose release, is the production of pharmaceutical pellets, more commonly known as
beads. Several methods can be employed in order to produce beads. For this study it was
opted to use a method, which has extensively been researched since the 1950s known as
extrusion-spheronisation.
Starches and starch based products have been utilised for many years as multifunctional
excipients in the production of solid oral dosage forms. For instance, starches have been used
as fillers, binders and disintegrants. The polymer rich matrix of a starch makes it highly versatile
in these applications. Furthermore, the low cost involved in manufacturing or sourcing starch
and starch based products, also makes it a commercially viable alternative to other market
available excipients which might be more expensive. Cassava is one of the world’s most
predominant sources of starch. It is globally grown and sourced in sub-tropic environments.
Being a sustainable product which produces a high yield of starch, this study investigated the
applicability of cassava starch as a filler in bead formulations using gliclazide as model drug.
13
Physical characteristics and flowability of cassava starch were evaluated with various methods,
which included thermo-analysis, moisture content, infrared spectrometry, and flow properties.
Beads were evaluated in order to determine whether extrusion-spheronisation improved the flow
of the starch. The physical characteristics such as friability, swelling and erosion, and
disintegration were also evaluated. Dissolution testing and analysis provided profiles which
were assessed and compared to a commercially available product, Diamicron®.
It was evident from the study that cassava is not the ideal filler to include in the manufacture of
beads, even though a single cassava bead formulation did provide prolonged release of the
drug over a 12 h period. Approximately 60% of the drug was pharmaceutically available within
the first 30 min of dissolution assessment and the remaining 40% dissolved slowly over the
remaining duration of the study. The dissolution profile obtained for this particular formulation
correlated with the arbitrary release profile of sustained drug release. It could therefore be
concluded that a product could indeed be produced which may be a viable candidate as a
commercially substitute for the current commercially available product, in terms of cost-
effectiveness and sustainability. From the study it was also evident that Avicel® provided a
better prolonged release profile in terms of mean dissolution time. Avicel® formulations proved
to render the most similar release profiles to that of the reference product, Diamicron®.
Keywords: Cassava, Starch, Extrusion-spheronisation, modified release, solid oral dosage
forms (SODFs), flowability, powder flow. Gliclazide, Avicel®, beads,
microcrystalline cellulose (MCC)
14
Uittreksel
Kassawestysel as ʼn vrystellings-modifiserende hulpstof in
vaste doseervorms met gliklasied as modelgeneesmiddel
Orale vaste doseervorms is die gewildste geneesmiddelafleweringssisteme wat kommersieel
beskikbaar is. Hierdie gewildheid kan toegeskryf word aan die gemak waarmee dit hanteer,
toegedien , en selfs vervoer word. Verskeie tipes orale vaste doseervorms is kommersieel
beskikbaar insluitend tablette, kapsules, meervoudige partikulêre sisteme en suigtablette.
Verskillende ontwerpe en vervaardigingsmetodes word gebruik in die bereiding van vaste
doseervorms ten einde verskillende tipes geneesmiddelvrystelling te verkry.
Geneesmiddelvrystelling is ʼn uiters belangrike oorweging tydens doseervormontwerp, veral vir
geneesmiddels met kort halfleeftye. Geneesmiddels met kort halfleeftye benodig gereelde
doserings op spesifieke tye. Meervoudige doseerskedules kan tot swak pasiënt-
meewerkendheid lei, wat kan lei tot een of meer oorgeslane dosisse. Swak
pasiëntmeewerkendheid veroorsaak ʼn afname in behandelingseffektiwiteit. Ten einde hierdie
struikelblokke te oorkom en geneesmiddelvrystelling te verbeter, word verskeie metodes
ingespan, onder andere, alternatiewe vervaardigingsmetodes en die gebruik van spesiale
hulpstowwe. ʼn Belowende vervaardigingsmetode wat tans baie navorsingsaandag ontvang om
verbeterde geneesmiddelvrystelling, hetsy dit verlengde, beheerde of meervoudige
dosisvrystelling behels, is die vervaardiging van farmaseutiese korrels, byvoorbeeld krale.
Verskeie vervaardigingsmetodes kan gebruik word om krale te vervaardig. In hierdie studie is
daar gebruik gemaak van ʼn metode wat sedert die 1950s breedvoerig nagevors is, naamlik
uitpers-sferonisering.
Stysels en styselgebaseerde produkte word al vir jare as multifunksionele hulpstowwe in die
vervaardiging van orale vaste doseervorms gebruik. So byvoorbeeld word stysel of stysel-
gebaseerde produkte onder andere as vulstof, bindmiddel en disintegreermiddel aangewend.
Die polimeerryke matriks verleen aan stysel die vermoë om as multifunksionele hulpstof gebruik
te word. Die lae vervaardigingskoste asook maklike verkryging van stysel en styselgebaseerde
produkte maak dit ʼn kommersieel aanvaarde alternatief as plaasvervanger vir duurder
hulpstowwe. Die Kassaweplant is een van die wêreld se mees algemene bronne van stysel.
Dit kom wêreldwyd voor in subtropiese gebiede. Omdat dit ʼn volhoubare bron is, wat ʼn hoë
15
stysel-opbrengs lewer, is daar in hierdie studie ondersoek ingestel na die bruikbaarheid van
Kassawestysel as vrystellingsmodifiserende hulpstof in die bereiding van krale.
Die fisiese eienskappe en vloeibaarheid van kassawestysel is gekarakteriseer met die gebruik
van verskeie metodes, waaronder termiese analise, voginhoudbepaling en infrarooi-
spektrometrie. Bereide krale is ook geëvalueer in terme van swelling, verbrokkeling,
disintegrasie en dissolusiegedrag.
Die resultate van die studie het getoon dat kassawestysel nie optimale krale gelewer het nie.
Ten spyte hiervan het ʼn enkele kassawe-kraalformulering verlengde vrystelling van gliklasied
oor ʼn 12 h tydperk getoon. Ongeveer 60% van die geneesmiddel is binne die eerste 30 min.
vrygestel, en die oorblywende 40% is in die oorblywende tyd van die studie vrygestel. Die
dissolusieprofiel het ooreengestem met die arbitrêre vrystellingsprofiel vir volhoude
geneesmiddelvrystelling. Vanuit die data kon gesien word dat die moontlikheid bestaan om ʼn
formulering te berei wat oor die potensiaal beskik om huidige kommersieel beskikbare produkte,
in terme van koste-effektiwiteit en volhoubaarheid, te vervang. Avicel® (mikrokristallyne
sellulose), - tans die standaard vir kraalbereiding — is ook in die studie gebruik om as maatstaf
vir kassawestysel te dien. Uit die resultate was dit duidelik dat Avicel® ʼn beter verlengde
vrystellingsprofiel verskaf het in terme van die gemiddelde dissolusie tyd. Avicel®-formulerings
het ook bewys dat die vrystellingsprofiel van die verwysingsproduk, Diamicron®, nageboots kan
word onder spesifieke eksperimentele toestande.
Sleutelwoorde: Kassawestysel, uitpers-sfeervorming, gemodifiseerde vrystelling, vaste orale
doseer vorme, dissolusie studies, vloeibaarheid, poeiervloei, gliklasied,
Avicel®, krale, mikrokristallyne sellulose.
16
Chapter 1
AIMS AND OBJECTIVES
1.1 AIM
The aim of this study was to investigate the possible application of cassava starch as an
excipient in a modified release solid oral dosage form. In conjunction with this investigation it
was also considered prudent to investigate the effects of using a multi-unit pellet (or
particulate) system as a modified release solid oral dosage form.
1.2 BACKGROUND
Changes in economies and socio-economic diversity as a result of globalisation and the growth
in consumerism have had both advantageous and disadvantageous consequences; e.g.
improved transport infrastructure, communication systems, energy generations, increased
health risks and increased levels of unemployment (Reddy et al., 2006:1-9; Storper 2000:
107-114). This is especially evident on the African continent. An area of concern, however, not
only on the sub-Saharan African continent, but also in developed economies such as Europe,
Japan and Northern America, is lifestyle dependent health risks. Lifestyle dependent health
risks in developed nations are a result of increased consumerism and an ever decreasing labour
intensive lifestyle (Badawi et al., 2004:76), whereas sub-Saharan Africa and other global
counterparts have increased health risks as a result of limited or no access to sufficient
resources, e.g. medical personnel, medical equipment and medication. Consequences of
health risks include sexually transmitted infections, low infant mortality rate, low female health
care and even the escalation in lifestyle dependent health risks. The latter is brought forth not
only by urbanisation but also the impoverishment of economically unstable nations
(Addo et al., 2007:1013; Meyrowitsch et al., 2007:32).
One of the most common lifestyle dependent health concerns is certainly insufficient glycaemic
control (Zimmet et al., 2001:782). Hyper- and hypoglycaemia are two pathological
manifestations of insulin insufficiencies, brought on either by defects in insulin secretion or
desensitised tissue response to insulin and glucose levels. In this study; only hyperglycaemia
was addressed.
17
The most predominant disease characterised by hyperglycaemia is diabetes mellitus type 1
(DMT-1) and 2 (DMT-2). DMT-1 is noted as absolute insulin insufficiency which
characteristically manifests in younger individuals, brought on by auto-immune-like degradation
of the insulin producing beta-cells located within the pancreas. DMT-2 is of slower onset,
manifesting in older individuals, caused predominately by individual lifestyles. DMT-1 is
managed by the frequent subcutaneous administration of exogenous insulin, possibly in
conjunction with oral medication. In contrast, due to its origin, DMT-2 is mainly managed by
lifestyle changes. Followed by therapies which include oral anti-diabetic medication, as first
choice regime. If these measures are inefficient at addressing DMT-2 pathophysiology
subcutaneous insulin can be employed as add-on therapy (Delamater, 2006:71).
Solid oral dosage forms (SODFs) are preferred, not only in anti-diabetic therapy but also in
other treatment protocols. These dosage forms are easier to administer to conscious patients;
requires little to no organoleptic consideration; needs little aseptic handling; can easily be stored
and transported; and added increased patient compliance with a decreased dosing interval. In
contrast to all these advantages, several disadvantages are also present, which include
administration difficulties for younger children, comatose and unconscious patients, delayed
action before gastro-intestinal absorption, limited dose capacity per dosage, and limited physical
size range of the dosage form. For treatment of diseases, such as diabetes that requires
regular control and monitoring, it is prudent to design a user friendly dosage form with ideally,
no incompatibilities with the patient’s physiological, pathological and lifestyle needs. Due to a
lack of an idyllic setting, an ideal product is not possible; however, researchers have attempted
to design a near perfect product that might fulfill patient related expectations and requirements.
These include, but are not limited to the lower dose load, controlled release of the drug,
affordability, sustainability and versatility (Bardonnet et al., 2006:2).
Current oral anti-diabetic therapeutic regimens include biguanides, e.g. metformin;
sulphonylureas, e.g. gliclazide; and thiazolidinediones, e.g. rosiglitazone. Biguanides are
preferred as a first-line regimen in DMT-2, whereas sulphonylureas are the second class of
treatment in later stage diabetic patients. Due to patient lifestyles and psychologies, patients
rarely pick-up on early symptoms and dismiss pathologies attributed by other factors in their life
(Lebovitz, 1999:1339). As a result, a large portion of diabetics might seek medical attention at
such a stage that first-line therapy might be insufficient and second-line therapy needs to be
initiated to manage symptoms).
18
Sulfonylureas, effective in the treatment of DMT-2, have been used as anti-hyperglycaemic
therapy since the mid-1950s. For many years this class of active compounds has been one of
the pillars of oral anti-diabetic therapy. Gliclazide is a second-generation sulfonylurea, which
stimulates insulin secretion by closing ATP-sensitive potassium channels in pancreatic beta
cells. It is classified as a weak acidic compound that comprises a larger hydrophobic character
than first generation sulfonylureas. Gliclazide also shows a lower tendency to induce
hypoglycemic episodes in patients. According to Remko (2009:77) gliclazide’s hydrophobicity
makes its activity more effective over an extended time duration. However, it is well known that
insufficient solubility of active compounds may lead to reduced absorption (Dressman et al.,
1998:12). Remko (2009:77) stated that although the second generation sulfonylurea derivatives
(including gliclazide) wereslightly soluble (water solubility of 138.4 mg.l-1 for gliclazide at 25⁰C),
they did depict a fast absorption rate. Through formulating gliclazide into a controlled release
dosage form (once daily dose), it should be possible to extend its activity from a half-life of
approximately 11 h. Characteristically this would increase patient compliance due to fewer dose
intervals (Bartels et al., 2004:9; Remko et al. 2009:77; Vanderpoel et al., 2004:2073).
Starches, which were used in this study are characterised by bio-polymers that have multiple
applications such as fillers, binders and disintegrants in the pharmaceutical and
biopharmaceutical fields. Possible reasons for the use of starches are:
Cost-effectiveness of the starch,
The fact that they are renewable materials,
Available in large quantities,
Non-toxic,
Biocompatible, and
Biodegradable.
In the 1980s it was discovered that certain starches retain unique features that suggest their use
as an excipient for the manufacturing of controlled release SODFs. Due to their versatile
properties, it is possible to obtain quasi-zero-order pharmacokinetic profiles with a very simple
and cost-effective manufacturing process. Tablets produced from starches show low sensitivity
in their release profiles towards manufacturing conditions such as tableting pressure (Chitedze
et al., 2012:32; Lemieux et al., 2009:172; Lenaerts et al., 1991:43). Furthermore, high amylose
cross-linked starch matrix formulations can be manufactured by using conventional tableting
techniques. This kind of technology ranks among the most cost-effective means of
19
manufacturing controlled release dosage forms for orally administered active compounds
(Lenaerts et al., 1998:229).
Cassava is produced in Latin America, Southern Africa, China, United Arab Emirates and India.
Its standing as a source of starch is rapidly mounting, particularly due to its low price on the
world market when compared to starches from other sources. The potential use of cassava
starch as binder as well as a matrix for the development of edible films has previously been
considered (Chitedze et al., 2012:32; Famá et al., 2006:8; Famá et al., 2007:266). However,
little has been studied on its ability to act as a controlled release excipient in orally administered
formulations (Casas et al., 2010:72).
1.3 OBJECTIVES
In order to achieve the aims of this study, the following will be done:
1) Characterisation of cassava starch with regards to physical properties, powder flow
properties, particle size and morphology.
2) Formulation of beads containing cassava starch as excipient in varying concentrations
(gliclazide, a weak acidic active ingredient (pKa of 5.6) which is poorly water soluble, will
also be included as a model drug).
3) Evaluation of different bead formulations in terms of their physical properties and drug
release profiles.
4) Comparison of different bead formulations, in terms of drug release behaviour, to a
commercially available equivalent (Diamicron®, a readily available gliclazide solid oral
dosage form was selected for this study).
20
CHAPTER 2
LITERATURE STUDY
2.1 INTRODUCTION
According to the 2013 fact sheet published by the World Health Organisation (WHO), diabetes
is prevalent in approximately 347 million individuals worldwide. Furthermore, the WHO also
estimates that diabetes will be the 7th leading cause of death by 2030 (WHO, 2013). In the
United States alone, an estimated 17.5 million patients were living with diabetes in 2007 at an
estimated cost of US$218 billion (Dall et al., 2010:297). By the year 2000, the health cost
concerning diabetes in sub-Saharan Africa was an estimated US$67.03 billion, both directly and
indirectly to patients and economies within this region. In 2010 an estimated 12.1 million
patients were living with diabetes and an estimate of 23.9 million will be living with diabetes by
2030 in this region alone (Hall et al., 2011:1-2).
Diabetes mellitus is defined as a chronic disease characterised by insufficient glycaemic control,
either due to insufficient insulin production, as in the case of DMT-1, or
tissue-insensitivity and insufficient response to insulin, as in the case of DMT-2. For the
purpose of this study only DMT-2 will be highlighted (Lebovitz, 1999:1339-1340;
WHO, 2013).
In contrast to DMT-1, patients with DMT-2 are of an older demographic and have a slower rate
of onset. The leading cause for DMT-2 is lifestyle dependent factors e.g., insufficient
cardio-vascular exercise, obesity, stress and inappropriate diets. It should also be noted that
genetic and environmental factors contribute to the onset of DMT-2 (Lazar, 2005:374; Lebovitz,
1999:1339-1340).
DMT-2 is described as a dysregulation in insulin and glucose control due to cellular decay of
pancreatic beta-cells; this being a result of over stimulation of these particular cells.
Consequently, these cells are depleted, or completely desensitised to changes in blood glucose
levels. In regards to treatment, all depending on the stage of development, early use of oral
antidiabetic medications can be used to improve glycaemic control (Fowler, 2007:131).
21
These drugs include:
biguanides, e.g., metformin;
sulphonylureas, e.g., gliclazide, glibenclamide, glipizide;
meglitinides, e.g., repaglinide, mitiglinide;
d-phenylalanine derivatives, e.g., nateglinide;
thiazolidinediones, e.g., pioglitazone, rosiglitazone;
α-glucosidase inhibitors, e.g., acarbose, miglitol;
amylin analogues, e.g., pramlintide;
glucagon-like-polypeptide 1 (GLP-1) analogues, e.g., exenatide, liraglitide; and
dipeptidyl peptidase-4 inhibitors, e.g., sitagliptin, saxagliptin, vildagliptin.
Each of these oral drugs targets either the improvement of insulin secretion, or the improvement
of tissue-sensitivity to insulin. In advanced cases patients might require exogenous insulin
administration in conjunction with oral antidiabetic therapy (Katzung, 2009:737; Lebovitz,
1999:1339-1340; WHO, 2013).
The number of patients diagnosed with chronic and lifestyle diseases such as diabetes, has
increased drastically since the industrial revolution (Cordain et al., 2005:341-344). With the
industrial revolution came a more consumer focused economy. The mechanisation of several
industries, for example agriculture, has led to a less labour intensive economy. This paradigm
shift is dominant in developed economies, e.g. Europe, Japan and North America. Increased
consumerism and decreased physical exertion have led to a more obese population
(WPRO, 2007:1-27).
Obesity is a condition characterised by a higher than normal body mass index and an increase
in plasma lipids. The increase in lipids within tissues influences the metabolic nature of insulin,
credited to the mass number of lipids that needs to undergo lipolysis. This places a strain on
insulin production by the pancreatic beta-cells (Day & Bailey, 2011:55-57).
Patients in developing nations such as sub-Saharan Africa, South America and some South
Asian countries, lack basic health care, education and nutrition. These disparities are present
due to socio-economic, geopolitical and industrial factors (Duraiappah, 1998:2167-2176).
Education, healthcare and nutrition are respectively perceived as basic human rights. Due to
the disparity present in these nations, individuals are deprived of these basic rights (Kawachi
et al., 1997:1491-1498; Wagstaff, 2002:97-102). Insufficient nutrition, may lead to insulin
22
dysregulation. Instead of maintaining a normal metabolism of glucose and lipids, insulin
production begins to reduce and metabolise protein in the body as a source of energy. This
dysregulation of insulin homeostasis leads to malnutrition associated pancreatic beta-cell
degradation (Taksande et al., 2008:19).
On the other hand, insufficient healthcare or the lack thereof, leads to delayed or wrong
diagnosis. The patient does not receive primary care or education in regards to proper nutrition
and healthcare, for example the identification of symptoms associated with diabetes
(Motala & Ramaiya, 2010:9-36). Due to the disparities in primary health care, these patients’
insufficient diagnosis or delayed diagnosis, palliative care would be considered redundant and
costly. Patients, who do receive any type of treatment, are provided treatment at an
unsustainable cost. SODFs are considered less expensive than any other dosage form, but
even this can amount to unsustainable expenditure on healthcare (Jewesson, 1996:1; Lajoinie
et al., 2014:1088-1089).
2.1.1 TREATMENT OF TYPE 2-DIABETES
The current first line regimen for DMT-2 is oral antidiabetics. This includes biguanides
(metformin), whereas sulphonylureas are considered an add-on, or second line therapy in more
progressive patients (Lahiri, 2012:73; Mcculloch, 2014:1-2). In this study, formulation
strategies, mainly using sulphonylureas, will be the focus.
Sulphonylureas, a second line treatment for early and progressive DMT-2, was first discovered
in the 1950s, with tolbutamide, chlorpropamide, acetohexamide and tolazamide being the model
drugs. Current treatment available is mainly second generation sulphonylureas, for example
gliclazide, glibenclamide and glipizide (Rendell, 2004:1339).
Sulphonylureas’ mechanism of action is based on the closure of the adenosine-triphosphate
(ATP)-mediated potassium ion channels involved in the secretion of insulin by the beta-cells.
Closure of these channels lead to the exocytosis of insulin in response to an increased
concentration of blood plasma glucose. These channels are not completely closed which
prevents possible sulphonylurea induced inhibition at high plasma concentrations (Panten et al.,
1996:1; Rendell, 2004:1339).
23
2.1.1.1 Gliclazide
Gliclazide (figure 2.1) is poorly water soluble and is rapidly absorbed after oral administration. It
is an intermediate acting hyperglycaemic drug, has a plasma protein binding of approximately
96% and is predominantly metabolised by the hepatic system, making it readily susceptible to
presystemic metabolism. Peak plasma drug concentrations occur within 3 to 4 h after
administration and the drug has a half-life of approximately 12 h.
Figure 2.1: Chemical structure of gliclazide
Table 2.1 reflects the physicochemical characteristics of gliclazide. Gliclazide has proven to
lead to an increase in insulin secretion in long-term treatment regimens. Due to the efficacious
nature of gliclazide in improving insulin secretion, hypoglycaemia is a dominant side-effect and
can be worsened by several drugs, for example aspirin, sulphonamides and alcohol. Other
adverse effects include cardiac dysregulation, cholestatic jaundice, leucopenia, vomiting,
diarrhoea, thrombocytopenia purpura, weight gain, inhibition of alcohol dehydrogenase
enzymes; and even cutaneous symptoms, such as photosensitivity (Fowler, 2007:132).
Due to the short half-life of gliclazide, it is predominantly available as a twice daily dose
regimen. Multiple dosing intervals increase the complexity of patient compliance. In more
complex dosing regimens the possibility of missed doses become more prevalent. With multiple
dose intervals a patient requires a larger amount of units. The increase in the amount of units
needed, may result in an increase in the cost of therapy per patient (Kardas, 2005:722).
24
Table 2.1: Physicochemical properties of gliclazide (revised from Drugbank.ca and
ChemicalBook.com)
Characteristics Gliclazide
Chemical Formula C15H21N3O3S
Assay ≥ 98%
Form Powder
Colour White
Melting Point ± 163 - 169°C
Molecular Weight 323.411 g.mol-1
pKa (Basic medium) 1.38
pKa (Acidic medium) 4.07
LogP 2.6
Water solubility 1.9 x 10-01 g.l-1
Metabolism Hepatic,
less than 1% is excreted via the urine
Toxicity LD50 = 3000 mg.kg-1
By extending the rate of release, it is possible to extend the presence of the drug in the
circulation, resulting in less dosing intervals and dose units. Consequently, this leads to a
reduction in missed doses and improved adherence to regimes; which ultimately leads to
improved therapeutic outcomes (Kardas, 2005:722). Modified release SODFs are a possible
approach to counter these disadvantages. The rationale of modified SODFs is based on
prolonging the drug present in the blood plasma. By extending drug-plasma levels, it is possible
to reduce the number of doses a patient requires and thus, improves patient compliance.
25
2.2 SOLID ORAL DOSAGE FORMS
Solid oral dosage forms (SODFs) are perceived as the most dominant drug delivery system
(Jivraj et al., 2000:58; Perioli et al., 2012:621). Various advantages exist that promote the use
of SODFs. These include the following:
Durability during storage and transport.
Ease in physical handling.
Minimal aseptic handling.
Ease of oral administration (Zhang et al., 2003:372; Zhang et al., 2004:371-390).
In contrast to these advantages, several disadvantages can be identified, which include:
Complexation and agglomeration of the various excipients or substances, and
bio-molecules found within the body for example serum albumin.
Administration difficulties in children, comatose patients, and patients with underlying
pathologies for example tumours or constriction of the oesophagus, which in turn makes
it difficult for patients to ingest (Sastry et al., 2000:138; Schiele et al., 2013:937).
Certainly one of the dominant drawbacks of SODFs is the drug susceptibility to various
metabolic processes, as in the case of presystemic metabolism (Dresser et al., 2000:42-
43; Paine et al., 2006:880-881).
To counter the abovementioned disadvantages and improve patient compliance as well as
convenience, researchers and manufactures have attempted several methods to modify the
release of drugs by altering the mechanism whereby the drug is released. This is accomplished
by chemically changing the drug molecule itself or changing the excipients of the product.
Mechanism based modifications include erosion-, diffusion-, dissolution- and osmosis controlled
release mechanisms (Das et al., 2003:12, Patel et al., 2006:58). However, controlled release
mechanisms have their own drawback. In the case of controlled release, the predominant
drawback is dose-dumping. Dose-dumping is the premature release of a drug from the
controlled-release dosage form. This is contradictory to the base rationale for the development
of controlled-release dosage forms (Krajacic et al., 2003:70).
2.2.1 FORMULATION OF SOLID ORAL DOSAGE FORMS
The formulation of a SODF is an important process in providing an acceptable and usable
pharmaceutical product for patients. Formulation is the process by which different constituents
26
and processes needed to manufacture a SODF, is determined and optimised. In order to
manufacture a SODF for either conventional or modified release of a drug, a number of factors
need to be considered. These include the excipients and manufacturing method (Allen et al.,
2011:2-6).
2.2.1.1 Excipients used in formulations
SODFs, for example tablets, include several excipients in their formulation. Each type of
excipient is incorporated to impart various characteristics or properties to the formulation.
These excipients include fillers, binders, disintegrants, glidants, anti-adherents, etc. (Alderborn,
2007:449; Allen et al., 2011:225). Table 2.2 provides various examples of the different excipient
types which can be utilised for the formulation of SODFs.
Table 2.2: Excipient types and examples
Type of excipient Examples
Fillers
Simple fillers: Microcrystalline cellulose
(Avicel®), micro-fine cellulose, lactose, calcium
phosphate, sugar, dextrose, etc.
Compound Fillers: Avicel® and colloidal sillica,
Avicel® and lactose, Lactose and maize starch,
Lactose and polyvinylpyrrolidone (Kollidon®),
sugars, etc.
Binders Kollidon® 30, 50, 90, VA-64, etc.
Disintegrants Ac-Di-Sol®, Primojel®, Explotab®, Kollidon® CL,
starches (Sta-RX® 1500), etc.
Glidants Magnesium stearate, colloidal sillica, etc.
Acting as a carrier agent for the drug and other excipients, fillers account for the majority of the
dosage form’s weight and volume. This increase in mass and volume allows for a higher
degree of control in regards to handling the drug. It should be noted that in some formulations
where the amount of drug is large enough, the filler might be redundant (Allen et al., 2011:225).
27
Fillers should fulfil several requirements before they are eligible to be included in a formulation.
These requirements include:
be chemically inert,
non-hygroscopic,
have biopharmaceutical acceptable properties,
have good technical properties,
possess an acceptable taste, and
be cost effective (Alderborn, 2007:449, Allen et al., 2011:225).
Of all the available fillers, none fulfil all of these requirements simultaneously. Due to the
presence of large amounts of filling agent certain properties which include flow rate,
compressibility and porosity of the filler, might be of concern (Alderborn, 2007:449,
Allen et al., 2011:225).
After mixing of the drug with the chosen filler a binder can be added. Adherence of the
individual molecules is achieved by the binding agent’s inherent mechanism of action. Binders
have various mechanisms by which binding occurs, namely:
Overcoming the electrostatic and intermolecular forces,
liquid based bonding,
mechanical interlocking,
the formation of solid bridges between particles after the evaporation of liquids and
natural occurring adhesive and cohesive forces (Alderborn, 2007:452; Allen et al., 2011:225).
Disintegrants, on the other hand, are added to the powder mix to facilitate drug release from the
SODFs after oral administration. Several mechanisms of action are possible for disintegrants.
They are:
swelling of the particles;
electrostatic repulsive forces between the individual particles;
restoration of the particle shape after compression, and
exothermic reactions.
However, there are currently three main mechanisms of importance. The first mechanism is
based on tablet rupture caused by swelling of the individual particles of the disintegrant powder,
after exposure to moisture. Secondly, disintegration can be facilitated by increasing penetration
28
of moisture through capillary fissures within the outer layers, eventually resulting in
fragmentation of the tablet. The final mechanism by which tablet disintegration can occur is by
deformation of the powder particles; particles with a natural elasticity may return to its previous
shape (Alderborn, 2007:450-452).
Another excipient that can be included into a formulation is a glidant. Glidants are incorporated
in the powder mix to improve flow properties. A glidant’s mechanism of action is based on
lowering the shearing forces between individual particles or changing the electrostatic
interaction between these particles (Faldu & Zalavadiya, 2012:923-924). Sufficient flow is
necessary in direct compression of certain SODF manufacturing, e.g. tablets and multi-
unit pellet systems. Glidants are recommended, if not required, in direct compression, though it
has proven effective and advantageous in wet granulation as well and even mixtures meant for
extrusion-spheronisation of pharmaceutical pellets (Alderborn, 2007:452; Allen et al., 2011:226).
2.2.2 MANUFACTURING METHODS OF SOLID ORAL DOSAGE FORMS
Different manufacturing methods can be used to form a SODF from the aforementioned
constituents. These methods include wet granulation, dry granulation, direct compression and
even extrusion-spheronised beads. Each of these methods is used in different ways, all
depending on the desired outcome or the characteristics of the excipients and drug.
2.2.2.1 Wet granulation
Wet granulation is considered the most cost effective as well as one of the oldest known SODF
manufacturing methods. A homogenous mixture is wetted with a suitable wetting agent (e.g.
water). The moist mixture can be milled or granulated by a granulator in order to form granules.
Prior to tableting, the granules are sieved to homogenise the granule size and to break
agglomerates. The homogenous granules are compressed into a tablet or placed in a capsule.
Wet granulation has several advantages; these include the usability of fine powders, flexibility in
the amount of wetting agents used, and the mixing of powders which do not adhere to each
other. On the other hand, some of the disadvantages include weak cohesion if the wetting
agent dries and did not supply sufficient cohesion between powder particles; and possible
hydrolysis of the excipients or drug (Summers & Aulton, 2007: 412; Tousey, 2002:8-13).
29
2.2.2.2 Dry granulation
Dry granulation can be used when excipients are for example moisture sensitive. Again, a
homogenous powder mixture is prepared. Two distinct methods can be used to manufacture
SODFs from this method:
Heavy-duty compression of the mixture into a large tablet, and
Roller compression of the mixture between cylinders.
The resulting product is milled to break it into granules. These granules are sieved to form a
homogenous granule size range. Subsequently, the granules are either compressed, or
encapsulated. One of the most prominent advantages of dry granulation is the use of this
method in manufacturing tablets containing moisture sensitive drugs and/or excipients. In turn,
dry granulation is not suitable for fine or physically incompatible powders. Mechanically, this
method of manufacturing has a high level of machine noise (Summers & Aulton, 2007:412;
Tousey, 2002:8-13).
2.2.2.3 Direct compression
A modern method of SODF manufacturing is direct compression of excipient powders into a
single unit. A homogenous mixture of dry powders is introduced into a suitable die via a hopper.
Compression of the mixture occurs by applying force to the mixture present in the die. This
compression is achieved by an automated press and punch. Compression of this powder
causes deformity of the powder particles. In some cases, when the applied force is removed
and the tablet exits the die, the individual particles of the powder might return to its original
shape due to the elastic nature of some of the excipient particles, possibly resulting in capping
or lamination of the solid tablet. These defects decrease the strength and durability of the
tablet. Binders can influence the elastic nature of the powder particles and thus prevent
capping or lamination; maintaining the integrity of the tablet (Alderborn, 2007: 467-473; Tousey,
2002:8-13).
2.2.2.4 Extrusion-Spheronised pharmaceutical pellets
Extrusion-spheronised pellets (beads) are a modern type of SODFs. Beads are manufactured
by extruding a wetted mass of excipients through a perforated screen to form uniform sized
extrusion. These extrusions are then spheronised to uniformly sized and shaped beads by the
use of a multi-bowl spheroniser. These beads can be delivered individually, collectively or
incorporated in a larger unit, e.g. multi-unit pellet tablets or capsules. These individual units are
30
collectively referred to as multi-unit particulate systems. Multi-unit pellet systems have proven
useful and advantageous in modified release SODFs (Gandhi et al., 1999:161).
2.3 IMMEDIATE RELEASE COMPARED TO
MODIFIED RELEASE SOLID ORAL DOSAGE
FORMS
The basic rationale of any drug release from a SODF is to provide an adequate plasma
concentration of the drug. This level falls in a concentration range; ranging from the minimum
therapeutic concentration to the minimum toxic concentration and this range is known as the
therapeutic index. Any drug concentration below the therapeutic index is sub-therapeutic,
whereas any concentration above the index is toxic. Immediate release of a drug is identified by
an initial release of drug which peaks after a certain time (relatively short) has passed. After the
peak is reached, the concentration level drops. In order to maintain a suitable therapeutic
concentration of the drug, the next dose needs to be timed correctly in accordance with the drug
half-life. A disadvantage of note is that with immediate release, sub-therapeutic or a toxic level
of the drug is possible. To counter this, modified release dosage forms are continuously being
developed. The rationale of modified, sustained or controlled release dosage forms is to
provide a constant plasma drug concentration over a prolonged time period. This extension of
drug present in the blood plasma reduces the number of doses required to provide a therapeutic
drug concentration (Allen et al., 2011:258). This described rationale for immediate drug release
compared to modified drug release is illustrated in figure 2.2.
Figure 2.2: Graph comparing immediate and controlled modified drug release
31
2.3.1 TYPES OF IMMEDIATE RELEASE SOLID ORAL DOSAGE FORMS
2.3.1.1 Conventional release solid oral dosage forms
The release of a drug from a conventional release SODF is characterised by the
physicochemical properties of the drug and dosage form. Conventional release SODFs are
known to release a drug as it transits through the body. These dosage forms are basically
administered as a unit-dose, implying for example that one tablet contains a specified dose
amount of the active agent. The tablet is easily administered by means of oral intake. Once the
tablet enters the gastrointestinal tract, it is exposed to gastrointestinal fluid, enzymes and other
biological factors. Transit is necessary for the disintegration and dissolution of the tablet, which
releases the drug from the SODF. The fluid present in the body saturates the tablet and
saturation allows for the incorporated excipients to fracture the solid tablet into smaller pieces.
A decrease in particle size leads to an increase in the surface area exposed to the fluid
environment; this increase allows for an increased rate of disintegration. After disintegration of
the SODF to a smaller particle size range; the particles will undergo dissolution. Once in
solution, the active agent can cross the epithelium into the blood stream, depending on the
permeability of the drug. At this stage the drug travels along the circulatory system to the site of
action. Both disintegration and dissolution can be described as a rate limiting step in the
intended release of a drug. The rate of these different steps can be influenced by the various
manufacturing methods, the choice of excipients, or the formulation itself. Other factors can
influence these steps as well, such as biological, environmental and physicochemical factors,
for example particle size of the excipients and water solubility. Reasons in favour of
disintegrating tablets include ease of administration, predetermined dose size, and patient
compliance. However, several drawbacks prevent the use of this type of tablet, which include
comatose patients, pathologies of the ora-esophogeal tract, and young children or elderly
individuals with difficulties in swallowing (Allen et al., 2011:225-226; Sahoo, 2007:20-31).
2.3.1.2 Effervescent Tablets
Effervescent tablets are designed to include a higher amount of drug; and most importantly,
disintegrate and dissolve within a glass of water. Key ingredients in the design of effervescent
tablets are bicarbonates or carbonates, and citric and/or tartaric acid, which in combination form
part of the disintegration system. As the effervescent tablet is exposed to water, it starts to
permeate the tablet. This in turn causes a reaction between the carbonate and acid which
produces carbon dioxide. Release of carbon dioxide disintegrates and dissolves the tablet. The
32
solution that is formed allows for a rapid onset of action and rapid emptying of the
gastrointestinal tract. By dissolving the SODF into a solution, it is possible for patients with
underlying pathologies which limit the intake of other SODFs, to ingest the medication. Elderly
and young children are also benefitted by using this type of SODF. One of the limitations of this
delivery system, however, is the organoleptic properties of the SODF excipients present in
solution, particularly the flavour of the solution. Due to the large amount of ingestible liquid, it is
recommended that the dosage form contains a flavouring agent (Alderborn, 2002:412;
Alderborn, 2007:456; Allen et al., 2011:228).
2.3.1.3 Chewable Tablets
Some SODFs are mechanically crushed by means of chewing. This ensures the complete
disintegration of the SODF into smaller particles. Though it should be noted that dissolution
does not fully occur in the mouth, but still in the gastrointestinal tract, this acts as the
disintegration process needed for drug delivery within the gastro-intestinal tract. As a result, this
allows for a faster dissolution of the SODF, and thus faster absorption. Due to the prolonged
presence within the mouth, flavouring is yet again a concern. If the patients do not prefer the
flavour of the tablet after chewing, it will affect patient compliance negatively (Alderborn,
2002:412; Alderborn, 2007:456; Ansel et al., 2011:227; Siewert et al., 2003:3).
2.3.1.4 Sublingual and Buccal Tablets
Another SODF that releases the drug immediately is sublingual and/or buccal tablets.
Sublingual tablets are SODFs which dissolve under the tongue, whereas buccal tablets dissolve
on the inside of the cheek or under the lip. The anatomical locations were both sublingual and
buccal tablets function can be seen in figure 2.3. These SODFs are designed to dissolve in the
mouth and be absorbed through the oral mucosa. Once these SODFs dissolve in the mouth, it
should not be swallowed. Again, these dosage forms rely on organoleptic considerations,
especially flavouring. A disadvantage of sublingual and/or buccal tablets is the limited dosage
size, due to the limited absorption capacity of the oral mucosa (Alderborn, 2002:413; Alderborn,
2007:457; Allen et al., 2011:227).
33
Figure 2.3: Sublingual and buccal route of administration (intranet.tdmu.edu.ua)
2.3.1.5 Multi-layer tablets
The basic concept of conventional multi-layer tablets is based on the repeated compression of
multiple layers containing incompatible active ingredients. It is also an acceptable practice to
colour the various layers, resulting in a uniquely identifiable product (Alderborn, 2002:412;
Alderborn, 2007:456).
2.3.1.6 Lozenges
Lozenges are tablets designed to slowly dissolve in the mouth. They are designed to either
have a local or systemic effect. Once lozenges are in the mouth, the saliva supplies the
necessary fluid which induces dissolution of the tablet and release of the drug. These tablets
can, however, also act as a simple slow release dosage form (Alderborn, 2002:413; Alderborn,
2007:457).
2.3.2 MODIFIED RELEASE SOLID ORAL DOSAGE FORMS
The rational by which modified release SODFs function is based on prolonging the presence of
the active ingredient in the blood plasma. This extended time of the drug present in the blood
plasma improves patient compliance and therapeutic outcomes. This is achieved by lowering
the number of doses required for the patient to maintain a therapeutic drug concentration
(Siegel & Rathbone, 2012:19-20).
2.3.2.1 Coated tablets
An approach to modified release SODFs is the coating of disintegrating tablets. Various
methods of coating were developed for maximum patient convenience, including enteric,
34
gelatine and film coatings. Each coat is applied using a spaying-dry method. After spraying the
tablet with the coating, the product is dried. One of the main reasons for applying a coating is to
improve dosage forms resistance in low pH environments. This is advantageous in the case of
a drug which is pH sensitive or the location of drug absorption is based in an environment with a
high pH (e.g. the intestinal tract) (Alderborn, 2002:412, Alderborn, 2007:456, Ansel et al.,
2011:227, Das et al., 2003:14).
2.3.2.2 Diffusion-controlled tablets
Diffusion-controlled release SODFs rely on moisture permeating it with subsequent drug
release. Diffusion-controlled release dosage forms are divided into two types, namely, matrix
and membrane types. In order for this system to function properly, the dosage form needs to
remain intact while in transit through the gastrointestinal tract. Upon exposure to moisture the
dosage forms starts to release the drug from the matrix or membrane which encompasses the
drug. Depending on excipients and manufacturing process used to manufacture this particular
SODF, the rate of drug release can be augmented to prolong drug release (Uhrich et al.,
1999:3183-3189).
2.3.2.3 Dissolution-controlled tablets
Dissolution-controlled release relies on the dissolution of poorly water soluble salts of the active
agent, using a slowly dissolvable carrier or covering of the drug particles with a slowly dissolving
coating (Uhrich et al., 1999:3183-3189).
2.3.2.4 Erosion controlled tablets
These tablets are a single unit system consisting of a matrix based structure. The active agent
is dispersed throughout the matrix. As the matrix starts to dissolve, the active agent is released.
This erosion leads to a loss in tablet weight and a predictable release profile of the active agent
(Colombo et al., 2000:201-202; Dey et al., 2008:1069).
2.3.2.6 Osmosis controlled tablets
Osmosis controlled release is based on a difference in osmotic pressure between the interior
and exterior environment of the dosage form. A semi-permeable membrane is permeated by
moisture due to this osmotic pressure difference. The active ingredient within the dosage form
starts to dissolve and the resulting solution is then pumped out of the dosage form via a single
orifice or through a semi-permeable membrane. This transport is a convective transport
35
process. Several pump mechanisms can be utilised, which include the introduction of a swelling
layer that forces the solution out as the layer expands. Another method is that the solution itself
exhibits swelling properties. Each of these methods produces pressure, thus forcing the
solution through the orifice. Osmosis controlled systems can be manufactured as a single- or
multi-dose system (Dey et al, 2008:1069; Gupta et al, 2010:571-582). Figure 2.4 illustrates an
example of an osmotically controlled release tablet.
Figure 2.4: Example of an osmotically controlled release tablet
2.3.2.7 Multi-layer tablets
Multi-layer tablets contain layers composed of different drug concentrations per layer; or each
layer is compressed to various degrees of density and strength. In the case of varying drug
concentration, upon the disintegration and dissolution of each layer, a different amount of the
active ingredient is released at various stages during gastrointestinal transit. If the
concentration of the drug is constant throughout the layers, the density of each layer influences
the rate of disintegration and therefore prolongs the release of the drug from the solid dosage
form (Alderborn, 2002:412; Alderborn, 2007:456). These multi-layer tablets are made by
compression of an initial amount of powder mix which is introduced into the die. After
compression the die is filled again with another layer. As a result of the applied force, the first
layer is compressed more densely than with the first compression. This delivers a denser and
mechanically stronger first layer. A slight variation on this method is an initial high pressure
compression of the first layer and then followed by consequent layers where each layer has a
reduced compression force applied to the layer (Abdul & Poddar, 2004:160-161).
36
2.3.2.8 Multi-particulates
A contemporary approach to modified controlled release dosage forms is the use of
multi-particulate components, which is a system constituted out of smaller individual units with
identical characteristics and properties. Multi-particulates have many advantages which make
them a suitable choice for controlled release, namely:
improved gastric emptying;
easily adjustable dosing;
multi-phase release profiles;
improved flow properties;
decreased dust and powder waste;
decreased tendency for dose dumping to occur;
reduction in both the dose frequency and dose size;
uniform transit through the gastrointestinal tract;
lower tendency to gastrointestinal irritation;
reduced individual variations;
possible multi-drug combinations;
lowered tendency for side-effects;
cost effectiveness;
provide a targeted and controlled release and
a shorter lag time.
This system of a single unit is useful in the case where varying concentrations of a drug need to
be present in a single unit, either a tablet of capsule. Individual particles can be designed with
different concentrations. Another advantage of multi-particulates is that incompatible drugs can
be incorporated into a single unit. The multi-particulates or pellets after manufacturing can now
be directly compressed into a single unit-of-use; or the pellets can be incorporated into a
capsule as in the case of this study. Several methods of multi-particulate manufacturing exist
(Ganhdi et al., 1999:160-161; Khan et al., 2014:2137-2140; Vervaet et al., 1994:131-132; Young
et al., 2002:87-92). These include:
layering,
freeze pelletisation,
cryopelletisation,
hot-melt extrusion and
37
extrusion-spheronisation.
2.3.2.8.1 Layering
Layering or coating is based on deposition of successive layers of an active ingredient. These
layers are deposited on a core. This core can be a crystal, an inactive agent or granule (Hirjau
et al., 2011:210). The following figure (figure 2.5) provides a diagrammatic representation of the
layering process.
Figure 2.5: Layering process for pharmaceutical beads (revised from slideshare.net)
2.3.2.8.2 Freeze pelletisation
Freeze pelletisation on the other hand is a manufacturing method where spherical matrix pellets
containing the drug are produced. A molten droplet containing the drug and excipients is
introduced into a temperature regulated column containing an immiscible liquid. .The column
consists out of various temperature regions, ranging from -40°C to 100°C. The liquid chosen for
the process needs to have a lighter density then the droplet. This difference in density between
the liquid and droplet allows for a natural conveyance of the droplet through the liquid. As the
droplet “drops” down through the liquid, it moves through the various temperature regions,
consequently forming a solid pellet. Layer by layer the pellet continues to form until it enters a
low temperature region (0°C to -40°C), at which time the deposited layers start to freeze and
solidify. This is an inexpensive and easily reproducible method of manufacturing pellets,
depending on the variables (Cheboyina & O’Haver, 2004:98-102; Lavanya et al., 2011:1345).
38
2.3.2.8.3 Cryopellitisation
Pellets produced via cryopelletisation, is when a droplet of organic or aqueous liquids are
conveyed through a perforated plate in the presence of liquid nitrogen and a solid pellet is
formed. The shape of the pellets is determined by the distance between the perforated plate
and the nitrogen reservoir. Pellet size is determined by the diameter of the perforations present
in the plate (Gandhi & Baheti, 2013:1624; Lavanya et al., 2011:1344).
2.3.2.8.4 Hot-melt extrusion
Hot-melt extrusion is a solvent free method, ideal for drugs that are unstable in the presence of
moisture. Several processes are used to form pellets by means of hot-melt extrusion and these
processes are:
Plastisation or melting of a drug dispersed throughout a solid medium which acts as a
thermal carrier.
Use of an extruder in order to shape the molten content.
Spheronisation at high temperatures to form uniform spheres.
Solidification of spheres into the desired shape (Lavanya et al., 2011:1345; Patel et al.,
2010:81-82; Young et al., 2002:87-92).
2.3.2.8.5 Extrusion-spheronisation
Extrusion-spheronisation is a multi-phase method of manufacturing, first developed in the
1950s. First, a homogenous powder mixture is wetted with a suitable wetting agent; for
example water. The resulting wet mass is introduced into an extruder. Once introduced into the
extruder, the mass enters a chamber via a hopper. The chamber contains multiple cylinders
which rotate at pre-set rotations and a perforated screen. As the cylinders rotate the mass is
pushed against the screen. Due to the sheering forces and compression of the mass against
the perforated screen, it is extruded through the screen. Figure 2.6 provides an approximate
idea of how the extruder functions. The size of the extrusions is determined by the diameter of
the perforations present in the extrusion screen. Finally, spheronisation of the extruded material
in a spheroniser is conducted. The spheroniser consists of a multi-bowl chamber with an
attached friction plate. As the extrusion enters the bowl, the rotating friction disk and supplied
compressed air create a rotation of the extrusion mass in such a manner that the extrusions
break into smaller sizes and produce spherical pellets. The size and shape of the final pellets
are determined by the rate at which the spheroniser rotates (Newton et al., 1995:101; Vervaet
et al., 1995:136; Young et al., 2002:87-92). For the purpose of this study and the cost-effective
39
nature of this method it was opted to use extrusion-spheronisation as the method of choice for
the manufacturing of the beads.
Figure 2.6: Radial extruder (revised from spheronizer.com)
2.4 STARCH AS A VERSATILE EXCIPIENT
Starch has proven versatile and invaluable in dosage form design. Due to the flexible nature of
starch and its various applications in the pharmaceutical industry, it is essential to investigate its
application in the design of modified release dosage forms (Dumoulin et al., 1998:161-162;
Ispas-Szabo et al., 1999:163-165; Lenaert et al., 1998:225). Starch, which is a natural
occurring polymer, has a multitude of applications in the pharmaceutical industry; it may be
employed as a filling agent, binder, disintegrant or even as a glidant (Bayor et al., 2013:17). It
has become a necessity to investigate applications of renewable sources for excipients. Starch
is considered a viable candidate in improving the release profile of an active ingredient and
resulting therapeutic outcomes (Dumoulin et al., 1998:161-162; Ispas-Szabo et al., 1999:163-
165; Lenaert et al., 1998:225).
Two principal polymers, amylopectin and amylose present in starch make for an ideal candidate
in the design of controlled release dosage forms. Figure 2.7 shows a structural comparison of
the two distinct polymers. These two polymers form a robust polymer-matrix. An important
property of starch powders is its tendency to gelatinise when moistened. This proves useful in
designing a dissolution- or erosion-controlled release tablet. When the mass is introduced to a
moisture rich environment, it begins to expand as it absorbs the moisture. This occurs as the
branched polymers expand and moisture permeates the polymer-matrix. The mass becomes
gelatinous and forms a pseudo-suspension or matrix. As the starch travels through the
gastrointestinal tract, metabolic processes start to dissolve the mass; this dissolution of the
40
starch allows for release of drug particles from the polymer matrix. An initial dose of the active
ingredient is released when the matrix starts to dissolve (Mandal et al., 2009:1348). Continuous
dissolution of the mass and release allows for sustained release of the drug (Mandal et al.,
2009:1348). As an abundant source of starch, investigation has been warranted in the possible
application of refined cassava starch as modified release filler.
Figure 2.7: Molecular and macroscopic structure of amylose and amylopectin
(revised from voer.edu.net)
41
2.4.1 CASSAVA
The arrival of European explorers in the “new world” has meant the sporadic spread of fauna
and flora across the globe. This migration led to many new discoveries within the indigenous or
non-indigenous environments (NOAA, 2008). A good example of this is
Manihot eschulenta Crantz (figure 2.8). Manihot eschulenta Crantz, otherwise known as yuca,
tapioca or cassava, is a perennial root of the Euphorbiacea-family, native to tropical and sub-
subtropical climates as seen on the South-American, South-Asian and the sub-Saharan Africa
continent. Although native to humid climates, this root is quite adaptable to various
environments (FAO, 2013:6-7).
Figure 2.8: Illustration of cassava plant and root (revised form theglyptodon.com)
Cassava is also globally known as one of the main sources for starch with an estimated global
production of 290 x 106 ton in 2012 (FAO, 2013:6). As a source of energy, cassava proves to
be an invaluable staple in the diet of several developing nations. Due to the relative short
lifespan of post-harvested roots, the refined starch powder lengthens the lifespan of the starch.
The process of refinement ensures a thriving economy, not only for subsistent farmers, but also
42
for refinement centres and post-refinement trading. Refinement is also crucial in improving the
safety profile of cassava starch, due to the presence of cyanide in the cassava root (Fáma et al.,
2006:8; Fáma, et al., 2007:266).
As stated before, cassava starch contains two distinct glucose derived polymers, linear and
helical amylose as well as a short chain branch amylopectin, in ratios of 1:3 – 1:4 (Charles
et al., 2005:2718). Amylose content ranges between 15.0 – 25.0% (Charles et al., 2005:2117;
De Floor et al., 1998:62; Moorthy et al., 2002:560-562; Nuwamanya et al., 2010:1;
Rollande-Sabaté et al., 2012:161). The physicochemical compositions of the aforementioned
polymers form a robust matrix. This natural matrix influences several important aspects of the
starch’s physicochemical properties. The level of crystallinity is directly affected by the number
of hydrogen bonds. If the crystalline structure shows a high level of rigidity, it would indicate a
high number of hydrogen bonds. With a high level of crystallinity, the more robust matrix would
show a tendency to lower fluidity and adversely affect physicochemical properties (Huang et al.,
2007:133). Other properties are highlighted in both tables 2.3 and 2.4.
Tabel 2.3: Content Properties (Revised from Moorthy, 2002:560,561)
Properties Cassava
% Yield from H2O-NH3 extraction medium 21.80 ± 0.540
%Total amylose extracted 0.37 ± 0.010
% Moisture content 10 – 14
% Fibre/Ash Content 0.01 - 0.8
% Lipid content 0.01 - 1.54
% Phosphorus content 0.01 - 0.01
Colour White
Granule shape Round, truncated, cylindrical, oval, spherical,
compound
43
Granule size 3 - 43 µm
Table 2.4: Physicochemical properties of cassava variants (Revised from Moorthy, 2002:569)
Variant Granule
size
[µm]
Reducing
values
Amylose
content
Past*
temp
[°C]
Vis**
2% paste
Swelling
volume
[ml.g-1]
Sol***
[%]
M-4 5.4 - 35.1 1.8 0.530 60.7 58.0 30.5 22.8
Kalikal
an
5.4 - 40.5 1.8 0.550 63.70 58.0 38.8 24.8
H-1687 5.4 - 40.5 1.4 0.540 55.68 58.0 25.5 23.6
H-2304 5.4 - 43.2 1.4 0.525 52.68 55.0 30.5 24.8
H-226 5.4 - 43.2 1.8 0.500 55.66 56.0 33.8 27.8
H-97 5.4 - 43.2 1.2 0.535 58.70 55.0 30.5 17.2
H-165 8.1 - 48.6 1.6 0.505 52.65 54.0 37.8 27.2
*Pasting, **Viscosity, ***Solubility
Cassava starch has a variety of applications in more than one industry. In the textile industry it
is used in clothing dye. The pharmaceutical industry utilises it as a versatile excipient, for
example fillers. Starch is used in the adhesive, rubber and foam industry. In the paper industry,
cassava starch is also utilised to improve the colour and paper quality of paper stocks. Organic
sugars and acids can be derived from cassava starch. Fructose syrup and gelatine capsules
can also be produced using sugars prepared from cassava starch. Employing bioreactor
processes which incorporate Aspergillus awamori and Lactococcus lactalis spp. lactis, L-lactic
acid can be produced. Phytase production is also possible with cassava starch (Fao, 2005;
Tonukari, 2004:5-6).
As observed in table 2.4, swelling is an important characteristic of cassava starch. The
tendency of the polymers to swell when in contact with moisture is of noteworthy importance in
the possible manufacturing of modified SODFs. Being susceptible to digestive processes, the
mass is dissolved in the gastrointestinal tract (Beneke et al., 2009:2612-2614). Dissolution of
the mass releases the drug from the resulting gelatinous mass. Due to the availability and
44
inexpensive nature of cassava starch, it has been deemed a promising candidate in the pursuit
of a cost effective and a renewable excipient in the production of modified release SODFs
(Dumoulin et al., 1998:361-362; Lenaerts et al., 1998:233-234). Though the versatility and its
renewability promises cassava starch to be a viable candidate for pharmaceutical product
manufacturing, scrutiny regarding the patient safety e.g. allergies and toxicity, would need to be
investigated. For the purpose of this study, this line of enquiry was forgone.
2.5 SUMMARY
In this chapter diabetes was briefly discussed, as well as one of the most dominant second line
oral antidiabetic drugs, gliclazide. An overview of different factors necessary in the formulation
of either immediate or modified release SODFs was also provided. Furthermore, starch as a
versatile and matrix-rich excipient in SODFs, specifically cassava starch as an inexpensive and
renewable source of starch, was discussed. It is this rationale that warrants the evaluation of
cassava starch as a suitable excipient in modified SODFs. In chapter 3, the materials and
methodology employed for this study will be discussed.
45
CHAPTER 3
EXPERIMENTAL METHODS AND MATERIALS
3.1 INTRODUCTION
Any pharmaceutical dosage form, conventional or specialised, is formulated in order for a
patient to receive an effective drug dose. Appropriate design and formulation require
methodical understanding of the functional factors that affect the physicochemical
characteristics of the drug and excipients used, as well as the absorption of the drug. The drug
and excipients incorporated into the formulation have to be compatible in order to produce a
product that is stable, efficient, striking, easy to administer, and safe. Furthermore, formulation
of a solid oral dosage form usually necessitates accurate processing control of the powder
mixture to guarantee a homogeneously formed product. Numerous excipients are
gravimetrically added to form the bulk powder with which homogeneity is accomplished through
optimum mixing. Homogeneity during tablet manufacturing is also accomplished through the
correct process used to achieve good flow of the mixture into the tablet die. Extrusion-
spheronisation was used in this study to increase the bulk density and increase flowability of the
formulations (Aulton & Taylor, 2013:480; Shah & Mlodozeniec, 1977:1377). Thus, in order to
evaluate which formulation is most appropriate, it is of utmost importance to evaluate the above-
mentioned factors influencing design and formulation.
This chapter deals with the pharmaceutical excipients (materials) used in the various
formulations tested. Moreover, it describes the experimental procedures employed to determine
the effect of these excipients on the physical properties of the beads formulated as well as on
the dissolution profiles of the formulations.
3.2 MATERIALS
The pharmaceutical materials employed in this study, their respective batch numbers as well as
where these materials were sourced, are presented in table 3.1. All of the materials were of
analytical grade and were used as supplied.
46
Table 3.1: Pharmaceutical materials employed in the various formulations, batch numbers and suppliers
Materials Batch nr. Source
Gliclazide 100111302045 Bal Pharma, Ltd. Bengaluru, India
Cassava starch 169A-27-11-12 Meelunie, BV.
Amsterdam, Netherlands
Cassava starch
(Mbundumali-namwera) Donated Malawi
Consolidated Starch 23871 Warren Chem Specialities Cape Town, South Africa
Avicel® pH 200 M939C FMC International,
Wallingstown, Ireland
Kollidon® 30 8608522440 BASF, SE.
Ludwigshafen, Germany
Hydroxypromethylcellulose 11040 Shin-Etsu Chemical, Ltd.
Tokyo, Japan
Hydrochloric acid 44836 Saarchem, Ltd.
Krugersdorp, South Africa
Methanol L361202 VWR International, Ltd.
Poole, England
Ethanol 180914ET Rochelle Chemicals, Cc.
Johannesburg, South Africa
3.3 CHARACTERISATION OF CASSAVA STARCHES
The physical properties of powders have a significant effect on the flowability and tabletability of
formulations. The primary physical excipient properties of importance are moisture content,
particle size and particle size distribution. Other properties (which are derived from the primary
properties) include flowability, compactibility and compatibility. The properties that were
evaluated during this study are described in the following sections.
47
3.3.1 THERMOANALYTICAL CHARACTERISATION
The presence and distribution of moisture depend considerably on the chemical nature of a
certain material, its physical properties such as particle size and porosity; and on the ambient
relative humidity (RH), which determines the equilibrium moisture content (Garr & Rubinstein,
1992:187-192; Teunou et al., 1999:109-110). Moisture may have noteworthy effects on the
density of materials, flowability, binding characteristics, lubrication properties, compression,
surface tension, tablet tensile strength and tablet toughness (Teunou et al., 1999:109-110;
Viljoen et al., 2014:731-741).
Thermoanalytical characterisation of the cassava starch powders was conducted at various time
intervals (30; 60; 120; 240; 360 and 480 min) and at various temperatures
(25; 30; 40; and 50°C).
3.3.1.1 Differential scanning calorimetric (DSC) analysis
DSC is used to determine physical properties based on thermal transition. Thermal stability of a
substance at increasing temperatures and specific time intervals can be evaluated using
DSC-analysis (Roy et al., 2002:399-400).
A Shimadzu DSC-60A (Shimadzu Scientific Instruments, Shimadzu, Japan) instrument was
used to obtain DSC-spectra of the cassava starch samples. Approximately 2 mg of each
sample was weighed into aluminium pans. These pans were sealed with a lid; each lid was
crimped in place by using a Du Pont crimper. The lids where pierced to form a small pinhole in
order to elevate possible pressure build-up within the pans. A similar sealed, empty pan was
used as reference. DSC-spectra were obtained at a heating rate of 10°C.min-1 under a nitrogen
purge of 30 cm3.min-1. The individual spectra were determined up to a temperature of 300°C
(Lemmer et al., 2012:331; Viljoen et al., 2014:732).
3.3.1.2 Thermogravimetric analysis
Thermogravimetric analysis (TGA) is based on the change in weight of a sample at various
temperatures (Ko et al., 2014:155).
TGA was conducted on each starch sample at a temperature range of 0 - 300°C.
TGA thermograms were recorded with a Shimadzu DTG-60 instrument (Shimadzu, Kyoto,
Japan). The weight of each sample was approximately 5 - 8 mg and heating rates of 10°C.min-1
under nitrogen gas flow of 35 cm3.min-1 were used. The theoretical weight loss during the
48
different conditions for each sample was calculated (in percentage) and compared.
Equation 3.1 applies to stoichiometric reactions with just partial weight loss such as dehydration.
100%mMn
MΔG
0Gas
m
[3.1]
Where the percentage content (G) is calculated from the weight loss (Δm) and the initial sample
weight (m0). M is the molar mass of the sample tested; MGas is the molar mass of the gas
liberated and n is the number of molecules liberated per starting molecule (Lemmer et al.,
2012:331; Viljoen et al., 2014:732).
3.3.1.3 Karl-Fischer titration
Moisture content for each sample was determined with a Mettler DL 18 Karl-Fischer titrator
(Mettler Toledo International LLC, USA). The Karl-Fischer solution was calibrated against a
predetermined mass of water. An accurately weighed (250 mg) cassava starch sample was
added to ethanol which was neutralised with the Karl-Fischer solution beforehand in a titration
beaker. The mixture was magnetically stirred and titrated with the Karl-Fischer solution.
Experiments were conducted in duplicate and the percentage water (w/w) calculated as follow:
10050W1000
CBMMoisture %
[3.2]
Where M is the Karl-Fischer titrant volume (ml); B is the volume (ml) of Karl-Fischer titrant for
the blank; C is the calibration amount (mg H2O.ml-1 Karl-Fischer titrant) and W is the sample
weight in grams (Aucamp et al., 2013:20; Viljoen et al., 2014:732).
3.3.2 INFRARED (IR) ANALYSIS
Vibrational characteristics, e.g. stretching and bending of different molecular bindings can be
examined and identified with the use of infrared (IR) spectrometry. Compounds can even be
identified using these IR-spectra due to individual compounds having distinctive IR-spectra
(Chistian, 2004:469-472; de Kock, 2005:60).
IR-spectra of the cassava starches were recorded on a Nicolet Nexus 470 FT IR ESP
spectrometer (Thermo Fischer Scientific, Waltham, Massachusetts, USA) over a range of
4000 - 400 cm-1 using the potassium bromide (KBr) referencing technique. Small samples of
approximately 2 mg were collected at different temperatures and time intervals; and individually
49
mixed with 200 mg KBr (Merck, Darmstadt, Germany) prior to analyses. This analysis was
repeated (with identical parameters, excluding the KBr reference) with a Bruker® Alpha Platinum
FT-IR Spectrometer (Bruker®, Billerica, Massachusetts, USA) in order to provide spectra with a
higher resolution (Aucamp et al., 2013:20; Lemmer et al., 2012:331).
3.4 SOLID ORAL DOSAGE FORMS
SODFs are presently the most preferred dosage form globally. The preference for SODFs is
accredited to the many advantages, for example: improved patient compliance due to ease of
administration, ease of transport, long shelf life and cost effective manufacturing (Alderborn,
2007:455-456; Hirani et al., 2009:162; York, 2013:7-8). As described in chapter 2, SODFs
consist of various excipients and the active pharmaceutical ingredient (drug). Each of these
ingredients is included in a formula for the manufacturing of a specific SODF. These ingredients
comprise, but are not limited to; fillers, binders, glidants and disintegrants, and most importantly
the drug. The drug and the different excipients as well as varying amounts of each of these
constituents need to be incorporated into a basic formula in order to be able to manufacture a
SODF.
3.4.1 PREPARATION OF BEADS
Pharmaceutical pellets (beads) are a modern method of SODF manufacturing and have proven
useful in the application for modified release dosage forms as well as the improvement of drug
release and physicochemical characteristics for example flow or physicochemical compatibility
between different drugs (Gandhi et al., 1999:160-162, Vervaet et al., 1994:131). The table
(table 3.2) below represents the factors and levels required to design the necessary
experimental formulations.
Table 3.2: Variables and different levels of each variable as employed in this study.
Factor
Levels
0 1 2
Drug (Gliclazide) 5% (w/w) 10% (w/w) 15% (w/w)
Filler Avicel® PH 101 Cassava starch Not applicable
Binder: Kollidon® 30
0% (w/w) 3% (w/w) 5% (w/w)
50
Polymer: HPMC
0% (w/w) 5% (w/w) 10% (w/w)
Powder mixtures were prepared. The composition of these mixtures were determined using a
partial factorial design. The composition of these mixtures were noted in table 4.2. The filler,
drug and binder for each formulation were weighed (mixture weighing 100 g) and transferred to
a glass bottle. Each of these bottles was covered with Parafilm® before closure with a screw
cap. The powders were mixed using a Turbula®-mixer (Model T2C, W.A., Switzerland) at
69 rpm for 10 min. After mixing, each powder mixture was wetted using a 70:30 deionised
water and ethanol mixture. The wetted mass was mixed using a mortar and pestle. After each
5 ml volume of the wetting agent added, the mixture was blended with a blender (Model FP731
Multi-Pro, Kenwood®, South Africa) for approximately 2 min. This was repeated till the correct
consistency was acquired. Upon completion of the addition of the wetting agent, the wetted
mass was passed through an extruder (Caleva® Extruder 20, Sturnminster Newton, England),
with the roller speed set at 32 rpm. A 1 mm diameter perforated screen was employed during
the extrusion. The resulting extrudate was spheronised in a multi-bowl spheroniser at 3000 rpm
for 10 min (Caleva®, Sturnminster Newton, England) (Chinyemba, 2012:21; Mallepeddi et al.,
2010:54). After spheronisation, beads were formed and the beads were dried at 40°C for 24 hr.
Bead samples were sieved to provide a mono-dispersed size range.
3.4.1 MORPHOLOGY OF POWDER PARTICLES AND BEAD
FORMULATIONS
Morphology is defined as: “the study of the forms of things, in particular” (Oxford dictionary) and
with the investigation of powder, bead and tablet morphology, various macroscopic phenomena
can be observed (de Kock, 2005:62). Differences between powder formulations, especially in
terms of flowability, packing formation and compression can be explained through differences in
their morphology. It is therefore important to investigate particle shape and size in order to be
able to predict for example, powder flow and packing arrangement (Hancock et al., 2004:980;
Lavoie et al., 2002:892; Velasco et al., 1995:2385).
3.4.1.1 Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) was used to identify the particle shape and surface
structure of the different starches used and bead samples that were prepared in this study.
51
SEM analysis provides information on microscopic level to better understand the macroscopic
behaviour of a powder or bead formulation (de Kock, 2005:62).
Each starch and bead sample was fixed to an aluminium stub using double-sided conductive
carbon tape to a sampling tray and dusted with an inert gas. Samples were subsequently
sputter-coated with a mixture of gold:palladium (80:20) to form a layer of approximately 28 nm
on the surface of the samples. In order to investigate the internal morphology of the different
bead samples, one or more beads of each sample were cut in half with a scalpel under a
stereomicroscope and the internal structure of these beads were coated with the gold:palladium
coating (Marais et al., 2013:6742; Sungthongieen et al., 2004:149). An Eiko® ion coater
(model IB-2, Eiko Engineering, Tokyo, Japan) was used in all coating procedures and operated
under a vacuum higher than 0.06 Torr. A FEI Quanta® 250 Environmental Scanning Electron
Microscope with a Field Emission Gun (FEI©, Eindhoven, Netherlands) was used to study the
samples and displayed on a commercial computer (Frizon et al., 2013:534; Marais et al.,
2013:6742).
3.4.1.2 Particle size analysis
Particle size analysis offers essential information reflecting the mean particle size and particle
size distribution within a powder or bead formulation. Understanding these physical properties
of powders and beads enables the formulation scientist to explain observed behavioural
differences between powders, especially in terms of powder flowability (Horn, 2008:38).
Particle size analysis of the cassava starch samples was conducted with a
Malvern® Mastersizer® 2000 instrument fitted with a Hydro 2000SM small volume dispersion unit
(Malvern® Instruments, Malvern, UK). The Hydro 2000SM dispersion unit was employed during
the particle size analysis of the raw material samples. For particle size determination of the
bead formulations, the analysis was performed with a 2000MU dispersion unit fitted to the
Mastersizer® instrument. As dispersion medium for all samples (powder and bead samples),
absolute ethanol was used at a stirring rate of 1500 rpm. The small volume dispersion unit
(Hydro 2000SM) was filled with 100 ml absolute ethanol for the powder samples, whereas the
(Hydro 2000MU) dispersion unit for the bead formulations was filled with 500 ml absolute
ethanol. A background measurement was taken for all samples to compensate for electrical
interference as well as possible interference from the dispersion medium. Upon completion of
the background measurement, a sample of the appropriate material was added to the
dispersion unit. Samples of the beads were dispersed in 6 ml absolute ethanol prior to addition
52
to the small volume dispersion unit. A sufficient quantity of the sample was added to obtain an
obscuration of between 10 and 20%. After a suitable obscuration was obtained, the particle
size of each of the samples was measured. Each measurement consisted of 12000 sweeps.
The particle size and distribution of each sample were measured in triplicate and calculated with
Malvern® Software (Malvern® Instruments, Malvern, UK).
3.5 FLOW PROPERTIES
Flow performance is often best described by quantification of the flow process. Several
methods have been defined, either directly, using dynamic or kinetic methods, or indirectly,
normally by measurements conducted on static powder beds (Staniforth, 2002:601). This
section describes the different methods utilised to determine the flow properties of the various
starch samples and bead formulations. These include the critical orifice diameter, flow rate,
angle of repose, powder density and compressibility.
3.5.1 CRITICAL ORIFICE DIAMETER
Critical orifice diameter (COD) is defined as the smallest orifice through which a powder will flow
freely without the application of any external aid or interference. The apparatus, developed by
Buys and co-workers (2005:40-42) was used to determine the COD of the powders (figure 3.1).
A set of copper rings (between 5 and 10 mm thick) with a centrally located orifice was used to
determine the critical orifice diameter. By placing the copper rings in increasing size on top of
one another, a tapered cone was formed. Each ring has a different size opening and the orifice
of each disc was machined to a set angle. The largest disc opening was 32 mm and the
smallest was 1.5 mm. A stainless steel hopper was fitted to the top of the funnel to create a
holding chamber for the powder. This set was placed on top of a three legged stand to a height
of 95 mm.
53
Figure 3.1: Apparatus used for the critical orifice diameter determination
A powder or bead formulation mass of 100 g was gently poured into the holding cylinder, while
the opening on the bottom ring was kept shut. Opening the bottom orifice resulted in the
discharge of the powder or bead formulation (if possible) from the holding chamber.
Interchanging the stacked rings allowed for changing the bottom orifice diameter, whilst keeping
the slope of the funnel constant until the smallest diameter was found through which each
powder could flow freely. This specific diameter was noted as the COD. Each study for both
original and dried powder, as well as for each bead formulation, was done in triplicate and the
average COD, standard deviation (SD) and percentage relative standard deviations (%RSD)
were calculated (Buys, 2005:40-42; Lambrechts, 2008:40).
3.5.2 FLOW RATE
The most direct method of assessing powder flow properties is the hopper flow rate. This
method describes the amount of powder that could be discharged through a funnel in a specific
time unit; normally per second (de Kock, 2005:64).
In order to determine the flow rate of the powders (original and dried samples) and bead
formulations, a stainless steel hopper with a diameter of 30 mm was used. A hopper, fitted with
a closed shutter at the bottom and which was raised 100 mm above the work surface, was filled
with a predetermined amount of powder or beads (approximately 100 g).
Subsequently, the shutter was opened and the time required to complete the discharge of the
powder mass was recorded (Lavoie et al., 2002:887-893). By dividing the powder weight with
54
the time recorded, a flow rate for the specific sample was calculated (using either equation 3.3
or 3.4). The procedure was performed in triplicate using different samples (100 g each) and the
average flow rate (g.sec-1), SD and %RSD were calculated (Sonnekus, 2008:23).
T
MF [3.3]
T
VF [3.4]
Where F represents the flow rate in g.sec-1; t represents time (sec). Mass (in g) is represented
as M and volume (in ml) as V.
3.5.4 ANGLE OF REPOSE
Angle of repose (AoR) is defined as a dynamic and static angle at which a powder comes to rest
when discharged from a container (Geldart, et al., 2006:104). Interactions between cohesive
and free-flowing powders can influence the flowability of powders. A higher angle indicates a
greater cohesive powder, whereas a lower angle is suggestive of a less cohesive powder
(Staniforth et al., 2007:170; BP, 2015: XVII N). The following table (table 3.3) provides the
quality of flow respective to possible angles at which a powder may come to rest.
Table 3.3: Flow quality of powders for various angles of repose (revised from Wells et al.,
2007:356)
Flow quality Angle of repose (degrees)
Excellent < 20
Good 20 - 30
Acceptable 30 - 34
Very poor > 40
After the complete discharge of the powder and bead formulations from the hopper, the height
and diameter of the resulting heap were measured and recorded (Martin et al., 1993:447;
Staniforth, 2002:207; Wong, 2002:2636). Figure 3.2 below represents these factors and their
respective dimensions in regard to the resting heap. Each experiment was done in triplicate.
Using the obtained data, the angle of repose was calculated using the following equation:
55
r
hTanθ [3.5]
Where h represents the height (mm) of the powder cone and r (mm) is the radius of the base
cone.
Figure 3.2: Angle of repose of a resting powder heap (bed)
3.5.4 POWDER DENSITY
Another characteristic of powders that influence powder flow which merits consideration, is the
density of a powder or formulation. The density of matter, including powders and beads, is
described as the mass of that matter divided by the volume that amount of matter may displace.
The flow of powder is affected by the density of a powder. Another property directly influenced
by the density of powders, is the compressibility of a powder. More densely powders tend to
have a weaker flowability, whereas a less dense powder tends to flow more freely (Jallo et al.,
2012:213; Traina et al., 2013:843).
The bulk and tapped densities of the powders and bead formulations were determined by
pouring 100 g of cassava starch or bead formulation into a graduated measuring cylinder. The
initial occupied volume was measured and the filled cylinder was placed on an
Erweka® Tapped Density Tester SVM 12/221 (Heusenstamm, Germany), which was set at an
amplitude of 5 A. Each sample was vibrated until a constant volume was obtained (BP,
2015:XVII A). Powder densities were calculated by the following equations:
b
bV
mρ [3.6]
56
p
pV
mρ [3.7]
t
tV
mρ [3.8]
Bulk density (ρb) was calculated as the ratio of the mass (m) to the initial (bulk) volume (Vb).
Similarly, the tapped density (ρt) was calculated as the ratio of mass to the final (tapped volume,
Vt) volume of the sample.
3.5.5 COMPRESSIBILITY
Carr’s index (percentage compressibility) and the Hausner ratio were respectively calculated
from the calculated powder and bead formulation densities. This provided a better
understanding of the compressibility of the starch powders and bead formulations
(BP, 201:XVII N, Jallo et al., 2012:216; Traina et al., 2013:843). Table 3.4 reflects the flow
quality corresponding to different values of Carr’s index and Hausner’s ratio. Equation 3.9 and
3.10, were employed to determine the compressibility.
t
bt
ρ
ρρ(CI) Index sCarr'
[3.9]
b
t
ρ
ρRatio Hausner [3.10]
Table 3.4: Flow quality as indicated by Carr’s index and the Hausner ratio (revised from
Aulton & Wells, 2002:134)
Flow Quality Carr’s Index (%) Hausner Ratio
Excellent (free flowing) 5 - 15 1.05 - 1.18
Good 15 - 18 1.18 - 1.22
Fair 18 - 21 1.22 - 1.27
Acceptable 23 - 28 1.27 - 1.39
Poor 28 - 35 1.39 - 1.54
Very poor 35 - 38 1.54 - 1.61
57
Extremely poor (cohesive) > 40 > 1.61
3.6 EVALUATION OF THE BEAD FORMULATIONS
3.6.1 FRIABILITY
Durability is another consideration of importance for all SODFs. Mechanical and physical
resilience are required during transport and handling. In order to determine the durability of
SODFs, the friability was determined. This can be simulated by a tumbling motion of a SODF in
a friabilator (Allen et al., 2011:233).
Friability was measured using an Erweka® Friabilator (Type TAR 220, Heusenstamm,
Germany). As described by the BP (2015), a bead sample of approximately 3 g from each
formulation, which was dusted and weighed beforehand, was loaded into the friabilator; 10 glass
beads were added to this sample. The initial weight was recorded as W0. The apparatus was
run for a total of 100 revolutions; at 25 rpm for 4 min, followed by the removal of the sample.
These samples were dusted and weighed again (W1) (BP, 2015:XVII G), and the percentage
friability was calculated for each formulation using equation 3.11. Each sample was evaluated
in triplicate.
x100W
WWFriability %
0
10 [3.11]
3.6.2 SWELLING AND MASS LOSS
Swelling was evaluated per published method (Singh et al., 2009:1123). A bead sample of
approximately 250 mg was evaluated for each formulation where the initial weight (W0) was
recorded beforehand. Each sample was placed in a basket and introduced into a USP type II
dissolution apparatus at 37 ± 0.5°C. The dissolution medium used was 675 ml of a 0.1 M
hydrochloric acid (HCl) solution for the first 2 h. Subsequently 225 ml of a 0.2 M phosphate
buffer was added and the pH adjusted to 6.8. Samples were drawn at predetermined time
intervals of 30, 60, 90, 120, 180, 360, 480, 600 and 720 min, blotted with filter paper and
weighed. The measurement of the swollen weight (W1) was noted. In order to determine the
loss (erosion) of the matrix, the swollen samples were dried in a regulated oven at 40 ± 05°C for
12 h and weighed afterwards to record the mass after erosion (W2). Percentage swelling as
well as percentage erosion was calculated with the following equations:
58
x100W
WSwelling %
0
1 [3.12]
0
21
W
xWWErosion % [3.13]
3.6.3 DISINTEGRATION
Disintegration of SODFs is recognised by the loss of its initial size as a result of the initial unit
breaking into smaller pieces. As described in chapter 2, the SODF is fragmented into smaller
particles; this in turn increases the surface area of the unit, thereby increasing the rate of
dissolution. Disintegration is a constant process (Ashford, 2007:300).
Empty capsules (size 0) were weighed. Each capsule was subsequently filled with beads and
weighed again. Six capsules containing spheronised beads were evaluated for disintegration.
This was evaluated using a disintegration tester (Erweka® Type ZT 323, Heusenstamm,
Germany). Distilled water was used as the disintegration medium and maintained at 37 ± 0.5°C
with a thermostat. The encapsulated beads were placed in baskets attached to the
disintegration tester, these baskets were dropped into the medium and then raised out of the
medium and this process was repeated until the capsules dissolved. The time it took for each
capsule to disintegrate was recorded (BP, 2015: XVII A).
3.6.4 ULTRAVIOLET-SPECTROPHOTOMETRIC ANALYSIS
Analytical chemistry is the field which encompasses the many methods relevant to chemical
analysis and quantification. Various methods can be employed in order to determine the
chemical composition or chemical presence of unknown substances, these include:
spectrometric, titrimetric and chromatographic methods (Krull et al., 2014:1-7). In the
pharmaceutical industry it is vital to determine the quality, composition and quantities of the drug
and other excipients present in dosage forms. One of the most widely used methods is
ultraviolet-spectrophotometry (UV-spectrophotometry). The science of UV-spectrophotometry is
based on the analysis of the energy transition that occurs when a compound is irradiated with
ultraviolet light. UV-spectrophotometry is a cost-effective analytical method which can be
utilised in order to determine the presence of a compound in solution, if a reference for
comparison for that specific compound is available. This rational can be used to determine the
concentration of a drug in solution, which in turn can be used to determine the dissolution
59
behaviour of a drug from a dosage form (Kassab et al., 2010:968-971, Krull et al., 2014:10;
Wilson et al., 2005:591-599).
3.6.4.1 Standard curve
A 25 mg gliclazide sample was vortexed in 10 ml methanol. The solution was added to 75 ml
methanol and placed in an ultrasonic bath (Labotec© EcoBath® model 103, Labotec©, Midrand,
South Africa) for 20 min. A standard solution of 250 ml was made by adding a 0.1 M
HCl-solution to the methanol mixture, which in turn was ultrasonicated for 20 min. The solution
was filtered with a 0.45 µm nylon membrane pre-filter attached to a syringe in order to remove
contaminants. The resulting filtrate wasultrasonicated for another 20 min. A concentration
range of 2 - 40 µg.ml-1 was prepared. These concentrations were acquired by adding 5 ml of
the stock solution to a 250 ml volumetric flask; 10 ml to a 100 ml flaks; 20 ml to a 100 ml flask;
15 ml to a 50 ml flask and 20 ml to a 50 ml flask. Each sample was made up to volume with
deionised water. A spectral analysis at 229 nm was conducted using an Analytikjena® UV-
spectrophotometer (Speccord® 200 Plus, Jena, Germany). The absorbance values obtained
from these analyses where used to determine if a linear relationship exists between the various
concentrations within the range. In order to determine the precision of this method, inter- and
intraday validations were conducted (Kassab et al., 2010:986-971, Jamadar et al., 2011:339).
3.6.4.1.1 Interday precision
Using the method described in 3.6.4.1 an analysis for linear regression was conducted in
triplicate on the same day to determine the precision of the range used, with a resulting %RSD
of less than 5% (Chinyemba, 2012: 27-29; Marais, 2013:98-101).
3.6.4.1.2 Intraday precision
For intraday variance the method described above was repeated on three consequent days.
%RSD must be less than 5% (Chinyemba, 2012: 27-29; Marais, 2013:98-101).
3.6.5 DISSOLUTION BEHAVIOUR
The dissolution behaviour of pharmaceutical dosage forms is important with regards to
determining the release profile of the drug from the dosage form. By comparing the dissolution
behaviour and release of a commercial SODF (Diamicron®) to that of an experimental SODF, it
is possible to determine the viability of the experimental SODF as a candidate for modified
release SODFs.
60
3.6.5.1 Assay
In order to determine the drug loading capacity of the beads, a 100 mg bead sample from each
formulation was crushed using a mortar and pestle. It was dispersed in 100 ml ethanol, stirred
for 12 h and sonicated in a Labotec EcoBath® (Model 103, Labotec©, South Africa) for 30 min.
The subsequent suspension was filtered through a 0.45 µm membrane filter. A 3 ml sample
was pipetted into a 100 ml volumetric flask to obtain the correct dilution. This dilution was
analysed at a wavelength of 229 nm using a spectrophotometer (Speccord 200 Plus,
Analytikjena®, Germany) (Chinyemba, 2012:45).
3.6.5.2 Dissolution studies
Dissolution studies were conducted using a USP paddle method in a six station dissolution
apparatus (Distek® 2500 dissolution apparatus, USA). For the first two hours 675 ml of
0.1 M HCl was used as the dissolution medium. Thereafter the pH was adjusted to pH 6.8 by
adding 225 ml phosphate buffer (pH 6.8). The stirring rate was set at 50 rpm and the
temperature maintained at 37 ± 0.5°C. Samples of approximately 5 ml were withdrawn using an
auto sampler (Distek® evolution 4300, USA) at predetermined time intervals of 0, 2.5, 5, 7.5, 15,
30, 60, 90, 120, 180, 240, 360, 480, 600, 720 and 1440 min. A sample was withdrawn at the
24 h interval; the stirring rate was adjusted to 250 rpm for a further 15 min and the last sample
was collected (BP, 2015:XII B; Singh et al., 2009:1123; USP, 2008:268-269). Samples of 3 ml
withdrawn at the various time intervals were diluted to a volume of 10 ml. The withdrawn
volume from the vessels, were replaced. This replacement medium was collected from a vessel
containing blank medium which was calibrated at each pH level and kept at the same
temperature as the experimental vessels. All withdrawn samples were analysed with a ultra-
violet (UV) spectrophotometer at 229 nm (BP, 2015: XII B; Singh et al., 2009:1123; USP,
2008:268-269).
3.7 STATISTICAL ANALYSIS
To compare the dissolution profiles, three statistical parameters were calculated. These were
the mean dissolution time (MDT), the dissimilarity factor (f1) and the similarity factor (f2). MDT is
the statistical moment of the cumulative dissolution process and is the mean time taken for the
drug to dissolve under in vitro conditions (Reppas & Nicolaides, 2000:231-232). MDT was
calculated with the following equation:
61
n
1i d
n
1i dmid
Δx
ΔxtMDT [3.14]
Where, MDT is the mean dissolution time in minutes, i the sample number, n the total number of
sampling times, tmid the midpoint between i and i-1 and Δxd the additional mass dissolved
between i and i-1 (Chinyemba, 2012:29; Marais, 2013:54-56)
Moore and Flanner (1996:64-74) used the similarity factor (f2) to compare dissolution profiles.
This factor compared the difference between the percentage drug dissolved per unit time for a
test and reference formulation. The value of the similarity factor is 100 when two dissolution
profiles are identical and approaches 0 as the dissimilarity increases. According to the
Food and Drug Administration (FDA), two dissolution profiles can be considered similar when f2
values between 50 and 100 were obtained (Costa et al., 2001:129). The following equations
can be used to calculate the dissimilarity factor (f1) and the similarity factor (f2)
100%
R
TR
fn
1t
t
n
1t
tt
1
[3.15]
100TRn
1150logf
0.52
1t
2
tt2
[3.16]
Where, f1 is the difference factor and f2, the similarity factor. Rt represents the assay time at
time t, Tt the test assay at the same time, n the number of pull points and wt the optional weight
factor.
3.8 SUMMARY
To determine the viability of this study, the previously described experimental methods were
utilised. It was the hope that through these methods a suitable formulation for modified release
could be produced in order to provide a modified release dosage form. The experimental
methodology was employed in such a manner to optimise a formulation containing suitable
excipients and concentrations of all the relevant ingredients, including the drug. The viability of
cassava starch as an excipient in modified release dosage forms was also determined using
62
these methods. In chapter 4 the results of these experimental methods will be given and
discussed in order to provide a clearly defined picture.
63
Chapter 4
EXPERIMENTAL RESULTS
4.1 INTRODUCTION
The determination of flow properties provides valuable information on powder mixtures intended
for the manufacturing of SODFs. Arguably, the most important properties that influence powder
flow are the size and morphology of powder particles. A range of parameters or properties can
be investigated to determine whether a powder exhibits acceptable flow. These parameters or
properties include compressibility, flow rate, angle of repose and critical orifice diameter, to
name a few. Another factor which could affect the quality of powder flow is the moisture content
of the powder. Moisture present in the powder can significantly diminish a powder’s flow quality
(Emery et al., 2009:409).
Physical characteristics of SODFs influence a product’s commercial suitability. These
characteristics include: friability, disintegration time and dissolution profile of a specific SODF.
Friability provides an indication of the robustness of the product during transport and handling.
Disintegration describes the process by which an SODF is broken down into smaller pieces and
as a consequence increase the surface area available for drug dissolution. Disintegration time,
therefore, is the time taken by the SODF to break up into smaller particles as specified by the
official pharmacopoeias (BP, 2015:XII A1). Dissolution profiles are used to determine the rate
and extent of drug release from a dosage form. Furthermore, dissolution data is useful in
determining the similarity or difference between respective formulations. Dissolution data may
also be employed to determine whether the release of a drug from a particular dosage form is
conventional or modified (Lourenҫo et al., 2013:367-368).
The different formulation variables and their levels were investigated by means of a fractional
factorial design (as discussed in Chapter 3). In order to provide a simplified method of
reference to the different formulations, it was decided to provide an identifier, as seen in
table 4.1. The identifier consists of a sequence of letters and numbers, representing excipients
and levels, for example, M.G5.5.5; where M/C is the filler; G5 is the drug and its concentration
(% w/w); the second 5 is the concentration (% w/w) of the binder and the final number (5)
represents the concentration (% w/w) of HPMC in the formulation.
64
Table 4.1 Identifiers for each successful formulation and the composition of each formulation
Identifier
Filler Gliclazide
concentration (% w/w)
Kollidon® 30 concentration
(% w/w)
HPMC concentration
(% w/w) Type Concentration
(%)
M.G5.3.5* Avicel® 87 5 3 5
M.G5.5.10 Avicel® 80 5 5 10
M.G10.5.5 Avicel® 80 10 5 5
M.G10.0.10 Avicel® 80 10 0 10
M.G15.0.5 Avicel® 80 15 0 5
M.G15.3.10 Avicel® 72 15 3 10
C.G5.5.5 Cassava 85 5 5 5
*M/C = Filler (M = Avicel® or C = Cassava), G5 = concentration (% w/w) of gliclazide, second
number (3) = concentration (% w/w) of Kollidon® 30, last number (5) = concentration (% w/w) of
HPMC
4.2 PHYSICAL CHARACTERISTICS OF CASSAVA
STARCH
Selected physicochemical properties of the cassava starches were evaluated accordingly to the
methodology as put forth in chapter 3, in order to decide which type of starch w could be used
as received or whether further processing was necessary. These properties tested included
relative humidity (RH) and moisture content. Infrared (IR)-spectrometry was employed to
determine if the two samples of starch were identical or represented different crystal forms of
the starch.
4.2.1 MOISTURE CONTENT AND THERMAL ANALYSIS
The average moisture content as determined by means of Karl-Fischer titrations of the
purchased and donated cassava starch is presented in figure 4.1. At time 0 min, the moisture
content for each sample was 15.82 ± 0.339% and 11.84 ± 0.156%, respectively.
65
4
6
8
10
12
14
16
0 30 60 120 240 360 480
Ave
% H
2O
Co
nte
nt
Time (min)
Donated
Purchased
Figure 4.1: Average moisture content of the donated and purchased Cassava starch,
at 40°C for various drying times
Powders that contain high levels (> 10%) of moisture have been associated with poor flowability
as well as poor powder characteristics (Crouter & Briens, 2014:70-73; Emery et al. 2009:414;
Nokhodchi, 2005:50). In order to determine whether the moisture was due to hygroscopicity or
a constituent of the polymer matrix, differential thermal (DT) and thermogravemetric (TG)
analyses were conducted. The thermograms are depicted in figures 4.2 and 4.3.
From these thermograms it was evident that weight loss, due to moisture evaporation, occurred
from the initial onset of heating, therefore indicating that the moisture present in the starch
samples was not part of the polymer matrix itself, but was present in the powder due to
hygroscopicity. Consequently, the temperature and time interval at which the moisture content
would be acceptable (6 – 10%) to provide conditions at which the flowability of the powder
would be acceptable, was determined (Crouter & Briens, 2014:70-73; Emery et al. 2009:414;
Nokhodchi, 2005:50).
After drying the starch samples at 25°C, 30°C and 40°C, over a time period of eight hours, the
moisture content was re-evaluated. It was determined that for the samples to show acceptable
flow, it should be dried at 40°C for 4h in order to achieve a moisture content of 6 – 10%. The
average moisture content of the samples dried at 40°C for 4h was measured at 8.07 ± 0.007%
and 8.79 ± 0.389%, respectively (Viljoen et al., 2014:730-742).
66
Figure 4.2: Thermogram of donated Cassava starch
Figure 4.3: Thermogram of purchased Cassava starch
67
4.2.2 INFRARED-SPECTROSCOPY
Figures 4.4 and 4.5 reflect the IR-spectra for both the donated and the purchased starch.
Figure 4.4: Overlay of IR-spectra for the donated (red) and purchased starch (black)
According to the IR-spectrum region of 1500 – 350 cm-1, the two samples of cassava starch
proved to be similar. This area serves as a fingerprint to determine, identify and compare
substances. Both samples contained a high amount of OH-groups and strong N-triple bonds.
In order to confirm the composition of the starch samples, it was opted to improve the resolution
of the IR-spectra. The resolution of the spectra was improved with the use of a Fourier
transform IR-spectrometry (FTIR). This improved resolution can be seen in figure 4.5.
Figure 4.5: IR-spectra from FTIR-analysis of the donated and purchased Cassava starch
68
FTIR provided a higher resolution and identification of the finger print region,
1500 – 350 cm-1, which indicated that the two starch samples were indeed related. However,
the improved resolution did indicate differences between the two samples. This proved that
both samples were of two unique starches (Vicentini et al., 2007:756-758). The finger print
region of the IR-spectra (obtained using FTIR-analysis) correlated with the IR-spectra given by
Huang et al. (2007:133).
4.3 PRELIMINARY EXPERIMENTS AND BEAD
MANUFACTURING
A 100 mg sample of purchased cassava starch was wetted with distilled water to determine if a
wet mass suitable for extrusion could be formed. The purchased starch was selected due to its
lower moisture content and possibly higher flowability. This mass needed to have a firm
consistency in order to be introduced into the extruder to obtain an acceptable extrudate. These
extrusions would be used in the manufacturing of beads. The first attempt proved difficult in
producing a mass of acceptable consistency for extrusion. A wet mass was produced with a
high concentration of water. This mass seeped through the perforations of the extrusion screen.
A mass of this consistency proved inefficient for the production of beads. Consequently, it was
decided to use a different wetting agent. An ethanol-water mixture was selected for wetting the
mass. With the addition of this wetting agent to another 100 g sample, a firmer wetted mass
was produced. This improvement in the consistency of the wetted mass correlates with the data
acquired by Millili and Schwartz., (1990:1411) who found that an ethanol:water mixture provided
a firmer consistency for extrusion-spheronisation. At 32 rpm, the radial extruder produced
extrusions of adequate consistency for spheronisation. After repeated adjustments with regards
to the rotation speed of the spheroniser as well as the duration of spheronisation, spherical
beads with irregular surface characteristics were obtained (Dhandapani et al., 2012:10-16; Joshi
et al., 2011:113). Kumar et al. (2012:1) stated that ideally beads should have a spherical shape
and a size range of approximately 600 – 1000 µm.
To improve bead quality with regards to size and shape, Kollidon® VA64 was added as binder.
The inclusion of Kollidon® VA64 was based on its use in matrix and tablet formulations
(Bhaskaran & Lakshmi, 2010:2431; Bühler, 2008:199-241). However, the mass that was
produced depicted a more viscous consistency which made extrusion difficult. It was decided to
investigate the the substitution of Kollidon® VA 64 with Kollidon , as an alternative binder.
Kollidon® 30 provided a mass with a less viscous nature; and consequently the extrusion-
69
spheronisation of the beads was successful. Beads produced from Kollidon® 30-containing
mixtures depicted a more spherical shape. The production of spherical beads prompted the
addition of gliclazide. A fractional factorial design (Table 3.2) was employed to investigate the
effects of excipients and concentrations on bead formulation.
Microcrystalline cellulose (Avicel®)was selected as alternative filler to cassava starch. Avicel®
is an industry standard for both direct compression and bead production (Dukic-Ott et al.,
2009:38-39; Vervaet et al., 2008:39). Smooth and spherical beads were successfully produced
with Avicel® containing formulations. After this production it was opted to improve the bead
spherocity by adding HPMC. HPMC was also selected for its application in matrix based
SODFs. However, Avicel® formulations containing no HPMC tended to produce irregularly
shaped and non-spherical beads, whereas all formulations containing HPMC rendered spherical
beads. HPMC has been described by Gandhi et al. (1999:166-168) as a recommended aid with
regard to bead manufacturing and bead quality. This could be attributed to the water solubility
and consequent gelling of HPMC, which is a low molecular weight polymer (Dukiƈ-Ott et al.,
2009:42-43; Gandhi et al., 1999:166-168). Cassava starch on the other hand, produced one
viable formulation that consisted of spherical beads. These beads therefore had a desirable
shape, size and mechanical strength. The success of the formulation, C.G5.5.5, could be
attributed to the respective concentrations of each excipient and the drug (Gandhi et al.,
1999:163-165; Khan et al., 2001, 350-354; Vervaet et al., 1995:136-143).
The aforementioned process in conjunction with the factorial design was used to determine
which mixtures would provide acceptable beads for the remainder of this study. These
formulations with acceptable quality is identifiable in table 4.1.
Table 4.2 provides the selected formulations and an indication of whether a successful
formulation could be manufactured from the selected combination of excipients.
Table 4.2: Selected formulations and respective excipients and concentration
Identifier Type of filler Filler
concentration
Drug
concentration
Kollidon
concentration
HPMC
concentration
Experimental
status*
M.G5.00 Avicel 95 5 0 0 Unsuccessful
C.G5.30 Cassava 92 5 3 0 Unsuccessful
M.G5.3.5 Avicel 87 5 3 5 Successful
C.G5.5.5 Cassava 85 5 5 5 Successful
M.G5.5.10 Avicel 80 5 5 10 Successful
C.G5.0.10 Cassava 85 5 0 10 Unsuccessful
M.G10.3.0 Avicel 87 10 3 0 Unsuccessful
C.G10.3.0 Cassava 86 10 3 0 Unsuccessful
M.G10.5.5 Avicel 80 10 5 5 Successful
C.G10.0.5 Cassava 85 10 0 5 Unsuccessful
M.G10.0.10 Avicel 80 10 0 10 Successful
C.G10.3.10 Cassava 77 10 3 10 Unsuccessful
M.G15.5.0 Avicel 80 15 5 0 Unsuccessful
C.G15.0.0 Cassava 75 15 0 0 Unsuccessful
M.15.0.5 Avicel 80 15 0 5 Successful
C.G15.3.5 Cassava 77 15 3 5 Unsuccessful
M.G15.3.10 Avicel 72 15 3 10 Successful
C.G15.5.10 Cassava 70 15 5 10 Unsuccessful
72
4.4 MORPHOLOGY AND SIZE
4.4.1 MORPHOLOGY
The morphology of the cassava starch powder particles was visualised by means of SEM, as
described in section 3.4.1.1 of this study. Both purchased and donated powders exhibited
spherical particles with occasional surface irregularities due to indentations (figure 4.6). From
these images it could be seen that both starch samples were in the same size range. It was
furthermore clear that the majority of the particles for both starches were less than 50 µm. As
poor flowability is usually observed for powders with an average particle size smaller than
100 µm, it would be expected that both starches will probably exhibit poor powder flow.
Additionally, agglomeration behaviour was observed in the micrograph depicting the particles of
the donated starch. Agglomeration behaviour is usually evident for small particles, indicating
cohesive behaviour which affects powder flow negatively (Kim et al. 2005:182-186; Landillon
et al., 2008:178-179, Lavanya et al., 2011:1338-1339; Staniforth & Aulton, 2007:169).
A B
Figure 4.6: Scanning electron microscopy micrographs of (A) purchased and (B) donated
starch
In figure 4.7 SEM-micrographs from the individual bead formulations are shown. For each
formulation, the bead shape, surface and internal structure are demonstrated, as indicated by
the letters A, B and C, respectively. Each micrograph in each (A, B and C) was conducted on
the same scales of magnification respective to that set. Set A had a scale of 1:500 µm, B a
scale of 1:20 µm and C a scale of 1:10 µm.
M.G5.3.5 M.G5.5.10 M.G10.5.5 M.G10.0.10 M.G15.0.5 M.G15.3.10 C.G5.5.5
A
B
C
Figure 4.7: SEM - micrographs of the different bead formulations (each set of three micrographs represents the following: A - full
view of the beads, B - the exterior surface morphology and C - the internal structure)
74
It was evident from figure 4.7 that a fairly spherical morphology was exhibited by the majority of
the formulations. The formulations, M.G5.3.5 and C.G5.5.5, each depicted a more spherical
shape whereas the remaining formulations portrayed either a bean (M.G10.5.5) or dumbbell
(M.G15.3.10) shapes that were indicative of insufficient spheronisation (Koester & Thommes,
2010: 1549-1550; Vervaert et al., 1995:136-141). These findings are in agreement with Chopra
et al. (2013:139), who stated irregularities in shape could occur with Avicel®-containing
formulations. The exterior surface morphology of each Avicel® formulation (figure 4.7 A) was
smooth and could be suggestive of good flowability (Kim et al. 2005:182-186; Lavanya et al.,
2011:1338-1339).
Avicel® formulation C.G5.5.5 depicted a general smooth surface, however, with closer
inspection of the exterior morphology (figure 4.7 B); a rough surface could be observed. This
formulation was the only formulation that contained cassava starch and considering the
micrograph of the cassava starch, the rough surface might have been attributed to the cassava
particles. The individual beads were grouped together with the assistance of a web-like matrix.
This matrix was formed due to the addition of the binder, HPMC. Both HPMC and Kollidon®
tend to form polymer matrices which influence the binding of excipients, as well as drug release
(Budiasih et al., 2014:54; Ingle et al., 2013:13; Mustafa et al., 2014:309-311).
In contrast to the cassava-containing formulation (C.G5.5.5), the individual particles of the
Avicel® filler could not be identified (figure 4.7 B) in the Avicel® bead formulations. The internal
structure of the Avicel®-containing beads (figure 4.7 C) tended to be more densely packed
together, whereas, C.G5.5.5 clearly showed cavities within the beads. These voids or cavities
could encourage moisture to penetrate the interior core of the beads, which possibly influenced
the dissolution rate of the drug (Yang et al., 2014:187-196).
75
4.4.2 SIZE DISTRIBUTION OF POWDER PARTICLES
Figure 4.8 represents the size-distribution of the individual powder particles and
beads.
0
150
300
450
600
750
900
1050
1200
1350
1500
1650
Size
(u
M)
Samples
d(0.1)
d(0.5)
d(0.9)
Figure 4.8: Size distribution histogram of both starches and bead formulations. Where
d(0.1) = 10% of particles smaller than, d(0.5) = 50% particles smaller than, and
d(0.9) = 90% of particles smaller than
Upon comparison of the particle size of both starch powders it could be postulated that the
starch powders are likely to exhibit poor powder flow. Lui et al (2008:109) stated that if the
mean particle size of material is smaller than 100 µm, the cohesive forces between the particles
would be higher. This could negatively affect the flow of the powders (Hart, 2015:2; Liu et al.,
2008:109; Staniforth & Aulton, 2007:170). From the size distribution of the bead formulations it
can be seen that they depicted an average median size range (d(0.5)) of
800 – 1000 µm, as illustrated in figure 4.8. From the particle size data it is expected that all the
bead formulations should exhibit good or acceptable flow as the d(0.9) values for all the bead
formulations were > 1000 µm. This indicated that 90% of the beads in the measured samples
were larger than 1000 µm. Additionally, from figure 4.8 a clear size difference can be seen
between the cassava starch powder particles and the C.G5.5.5 beads. This difference in size
could presumably improve the flowability of the product (Hart, 2015:2; Lui et al., 2008:109;
Vervaet et al., 1994:131-132). Size variation of the beads could be attributed to variation that is
seen in all pharmaceutical manufacturing processes. These processes include the amount of
76
wetting agent added and the duration of time after extrusion, before introduction into the
spheroniser (Mallipeddi et al., 2010:56-62; 2014:362-366).
4.5 FLOW PROPERTIES
Several parameters were used to describe the flow behaviour of the cassava powders and the
bead formulations. These parameters and the values obtained are reported in table 4.3.
Both starches presented a critical orifice diameter (COD) value of 16 mm, whereas all the bead
formulations depicted a COD value of 6 – 7 mm. A high COD value indicates poor powder flow
and a smaller COD value is an indication of improved flow. The improved flow was a
consequence of the enlarged size of the beads as described in section 4.4. No marked
differences were observed pertaining to the COD values for the individual bead formulations.
Neither flow rate nor angle of repose could be determined for both starches. These results
indicated weak powder flow which corroborated the postulation in section 4.4, i.e. that the small
size of the starch powder particles is expected to be detrimental to powder flow. The moisture
content (15.82 and 11.84%) of the donated starch and purchased starch may also aggerasvate
the poor flow behaviour. Although a marked improvement (in comparison to the starch
powders) in the flow rate was observed for all bead formulations, differences could be seen
concerning the different bead formulations. These differences can be attributed to the shape of
each formulation’s individual beads as depicted in figure 4.6 and 4.7. As the shape of the
different bead formulations is not perfectly spherical, contact between the beads might increase
providing more friction and thus impeding flow (Javadzadeh et al., 2015: 86-97; Korhonen et al.,
2000:1141; Zhang et al., 2003:6-7; Zhang et al., 2004:371-390).
Table 4.3: Flow properties of both starches and bead formulations
Critical orifice diameter
(mm)
Flow Rate (g.s-1)
Average angle of
repose (°)
Density (g.cm-3)
Compressibility
Bulk Tapped Carr’s Index (%) Hausner Ratio
Donated 16 ± 0.0 No flow No flow 0.6 ± 0.01 0.8 ± 0.02 0.3 ± 0.01 1.4±0.02
Purchased 16 ± 0.0 No flow No flow 0.5 ± 0.02 0.8 ± 0.03 0.3 ± 0.01 1.5±0.02
M.G5.3.5 6 ± 0.6 4.8 ± 0.23 26.6 ± 0.22 0.8 ± 0.05 0.9 ± 0.01 0.1 ± 0.06 1.2±0.09
M.G5.5.10 6 ± 0.0 4.7 ± 0.13 28.3 ± 2.59 0.8 ± 0.01 0.9 ± 0.00 0.1 ± 0.02 1.1±0.02
M.G10.5.5 6 ± 0.0 4.6 ± 0.12 25.7 ± 1.45 0.8 ±0.01 0.9 ± 0.01 0.1 ± 0.01 1.1±0.01
M.G10.0.10 7 ± 0.6 5.2 ± 0.15 28.3 ± 1.23 0.8 ± 0.01 0.9 ± 0.00 0.1 ± 0.01 1.1±0.01
M.G15.0.5 6 ± 0.0 3.8 ± 0.08 29.8 ± 2.24 0.8 ± 0.00 0.9 ± 0.00 0.0 ± 0.01 1.0±0.01
M.G15.3.10 6 ± 0.0 3.6 ± 0.0 30.1 ± 0.22 0.8 ± 0.01 0.9 ± 0.00 0.1 ± 0.11 1.1±0.01
C.G5.5.5 6 ± 0.0 4.9 ± 0.14 26.3 ± 0.55 0.8 ± 0.01 0.9 ± 0.00 0.1 ± 0.00 1.1±0.01
78
Despite the differences in flow rate, all bead formulations exhibited an approximate flow rate of
3.64 – 5.17 g.sec-1, indicating good flowability. Both starches and all the bead formulations
exhibited Carr’s indices and Hausner ratios of < 1 and 1.07 – 1.18, respectively, which is
indicative of excellent (free) flow.
The bulk density of 0.60 g.cm-3 and 0.55 g.cm-3 for the donated and purchased starches,
respectfully, were less than that of the beads, which ranged from 0.79 - 0.83 g.cm-3. Tapped
densities of both starches and beads ranged from 0.81 – 0.91 g.cm-3. These results indicated
that the powders packed more densely after tapping, which could be ascribed to the smaller
particles, leaving less void spaces after being rearranged. The beads on the other hand, did not
rearrange as compact as the powder due to the rigidity of the beads and their inability to fill the
void spaces in-between. Considering the data for the different flow parameters, it is evident that
all the parameters on powder flow for the different bead formulations indicated good to excellent
flow behaviour. This is to be expected as beads are known to exhibit acceptable to excellent
flow due to their size and more spherical nature. It is therefore clear that the formulation of
beads resulted in a pronounced improvement in the flow properties in comparison to the
flowability of the starch powders.
From the data it could be concluded that cassava starch does not have an acceptable flow
quality, which excludes it as an excipient for direct compression without modification or the
inclusion of a glidant. However, the cassava bead formulation exhibited the necessary
flowability and therefore potential to be introduced into a tablet press for compression into a
multi-unit pellet system. The data also corroborates the influence of size on flow quality, by
increasing the size of the particles, with the help of bead manufacturing, the flow quality
improved dramatically.
4.6 EVALUATION OF BEAD FORMULATIONS
Each bead formulation was subjected to various experiments in order to determine the viability
of the individual formulations for manufacturing as a SODF. In this study one of the objectives
was to attempt the compression of the beads into a single unit product. The compression of
these beads into a single tablet is known as a multi-unit pellet system(Reddy et al., 2012:42-54).
This method of SODF manufacturing is based on the concept of providing a modified release
dosage form or a fixed dose combination. Each of these has their respective rationale in
support of multi-unit pellet system manufacturing. Different types of multi-unit pellet system
could be manufactured including direct compressed multi-unit pellet system and encapsulated
79
particulates. One of the most researched types, are manufactured with direct compression of
particulates into a single tablet (Reddy et al., 2012:42-54). The rationale behind a multi-
unit pellet system compressed from beads was in part to produce a convenient product for drug
delivery. Samples of the bead formulations were introduced into a Korsch® XP1 tablet press.
The tablets produced from these beads proved difficult to produce. As each produced batch
ejected from the die they either crumbled as they left the die or as they were moved . This
could be attributed to insufficient mechanical strength of the compressed tablets. The
insufficient mechanical strength may be attributed to the hardness of the individual beads being
too high for the necessary deformation in order to compress into a single tablet, or insufficient
cohesion between the individual beads. Therefore, it was decided to encapsulate intact beads
in size 0 gelatine capsules to render a SODF.
4.6.1 FRIABILITY
Friability of the individual bead formulations provided information regarding the resistance of the
beads to breaking or splitting during handling and transport (Chapter 3). Table 4.4 presents the
average percentage friability results obtained for the different bead formulations.
Table 4.4: Percentage friability of bead formulations
Bead formulations Average % friability
M.G5.3.5 1.6 ± 3.22
M.G5.5.10 0.1 ± 0.27
M.G10.5.5 0
M.G10.0.10 0.7 ± 0.20
M.G15.0.5 0
M.G15.3.10 0
C.G.5.5.5 2.0 ± 0.88
Friability values could only be successfully determined for four formulations as the other
formulations depicted extremely brittle beads. The analysis indicated that C.G5.5.5 provided a
poor friability (2.0 ± 0.88); and M.G5.5.10 and M.G10.0.10 passed the 1% acceptance value
specified by the British Pharmacopoeia (BP, 2015: XVII G).
80
4.6.2 SWELLING AND MASS LOSS
During the swelling and mass loss study the results relating to the cassava starch beads were
not obtainable due to the disintegration of the beads. With the removal of the basket containing
the cassava beads, the residue of these beads seeped out of the basket, which consequently
made the determination of the weight impossible. This could be ascribed to the pH susceptible
nature of cassava starch. Cassava rapidly dissolves in a low pH or acidic medium. Figure 4.9
reflects the cumulative increase/decrease in mass (mg) measured for the Avicel® bead samples
per time interval (min).
A drastic increase in mass was observed during the first 30 min for all of the Avicel® bead
formulations, whereafter no substantial increase or decrease in mass for the following 90 min
could be observed. During the first 120 min, the beads were exposed to an acidic medium
whereafter the medium was changed to a more alkaline medium (pH 6.8). In the alkaline
medium (180 – 720 min) clear differences could be observed between the different Avicel®
formulations.
Low gliclazide content allowed for a higher degree of swelling and this could be attributed to the
ease at which the moisture permeated the beads due to the fact that less hydrophobic drug was
present that would be able to form a barrier against water penetration into the beads. The
formulations containing a higher concentration of gliclazide (10 or 15% w/w) depicted decreased
swelling. The higher drug content might have decreased the rate as well as quantity of liquid
that penetrated the beads due to its natural hydrophobic character (chapter 2). Moreover,
formulations comprising higher concentrations of HPMC in combination with a lower
concentration Kollidon® 30 portrayed a marked increase in the percentage swelling. This
increased swelling could be attributed to the polymer rich HPMC. HPMC is highly hydrophilic
and thus attracts moisture into the beads resulting in swelling/expansion of the matrix. The
swelling of the polymer could cause pores present in the beads to open and allow more
moisture into the beads (Akhgari et al., 2007:51-58; Ghori et al., 2014:1-17;Scholtz et al.,
2014:486-501; Viridén et al., 20010:60-67; Viridén et al., 2011:470-479).
0
200
400
600
800
1000
1200
1400
1600
1800
0 60 120 180 240 300 360 420 480 540 600 660 720 780
Cu
mu
lati
ve
Ma
ss
In
cre
as
/De
cre
as
e (
mg
)
Time (min)
M.G5.3.5
M.G5.5.10
M.G10.5.5
M.G10.0.10
M.G15.0.5
M.G15.3.10
Figure 4.9: Cumulative mass increase or decrease of Avicel® beads as a function of time (min) after exposure to calibrated pH
environments.
82
According to Goyal et al. (2009:95-96) swelling of HPMCcontaining formulations are pH
dependent. This was also evident within this study as the amount of swelling in the alkaline
medium increased noticeably relative to the amount of swelling in the acidic medium. The lower
the concentration of Kollidon®, the lower the amount of swelling and also the mass loss. After
the swelling experiment, the beads were dried for 12 h until no noticeable weight loss was
observed. Annexure C displays the results obtained after drying of the beads and from these
results it was evident that the loss on mass of the different Avicel® bead formulations all
averaged approximately 170 mg after drying, thus portraying an approximate loss in mass of
30% (Akhgari et al., 2007:51-58; Ghori et al., 2014:1-17; Viridén et al., 2011:470-479).
4.6.3 DISINTEGRATION
All capsules, irrespective of formulation disintegrated in less than 5 min, which complies with
specifications of the British Pharmacopoeia (2015: XVII S). Upon disintegration of the capsule
shells, the beads were dispersed throughout the disintegration medium.
4.6.4 DISSOLUTION BEHAVIOUR AND STATISTICAL ANALYSES
Several variables needed to be optimised in order to evaluate the bead formulations. This
included the amount of beads required to compare dissolution profiles with the control product,
i.e. Diamicron®, and standardisation of the analytical method that was employed.
4.6.4.1 Standard curve
A stock solution was prepared according to the method described section 3.6.4. The solution
consisted of 25 mg gliclazide dissolved in a 2:3 methanol:HCl - solution. This stock solution
was used to prepare a standard solution to construct a standard curve which was utilised for the
first 2 h of the dissolution study. Another stock solution was prepared with a
2:3 methanol and phosphate solution with a pH of 6.8. This solution was used to prepare a
standard solution in order to construct a standard curve that was used for the remainder of the
dissolution study (2 hr to 12 hr).
4.6.4.2 Linearity
The following figure (figure 4.10) provided a graph depicting a linear relation between the
absorbance and concentration of gliclazide in a methanol:HCl solution.
83
y = 0.0373x - 0.0021R² = 0.9997
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
0 5 10 15 20 25 30 35 40 45
Ab
so
rba
nc
e (
un
its
)
Concentration (mcg.ml-1)
Figure 4.10: Standard curve for gliclazide dissolved in 2:3 methanol:HCl solution
Standard solutions were prepared according to the method described in section 3.6.5. Linearity
was observed in a concentration range of 2 – 40 µg.ml-1, with a R2 - value of ≥ 0.999 for both the
acidic and alkaline media. At concentrations higher than 40 µg.ml-1 deviation from Beer’s law
was observed. This phenomenon was also noted by Ibragimova and Ikramov (2015:73-74) with
another sulphonylurea, glieipiride. Glimepiride and gliclazide are both examples of the same
drug classification, namely second generation sulphonylureas, which are chemically and
pharmacologically related (Kalra & Gupta, 2015: 101-104, Kalra et al., 2015:314-315).
4.6.4.2.1 Intra- and interday precision
Intra- and interday precision fell within acceptable limits as a %RSD value of ≤ 5% was obtained
for both intra- and interday precision (Chinyemba, 2012: 27-29; Marais, 2013:98-101).
4.6.4.3 Dissolution
Dissolution studies were conducted on samples from each formulation containing 30 mg
gliclazide. The sample weight was determined based on assay results obtained as per
described method (section 3.6.5.1). The selection of the 30 mg drug dose per bead sample was
based on the drug dose of the control (Diamicron® tablets; 30 mg gliclazide per tablet). The
dissolution profiles of the different bead formulations as well as the control are presented in
figure 4.11.
From the dissolution profiles it is observed that the profiles of two formulations appear differently
from the other profiles, namely, formulation C.G5.5.5 and M.G15.3.10. From figure 4.11, it is
evident that C.G5.5.5 exhibited a burst release of drug within 15 min of the study.
0
20
40
60
80
100
120
0 60 120 180 240 300 360 420 480 540 600 660 720 780
Perc
en
tag
e D
isso
luti
on
(%
)
Time (min)
M.G5.3.5
C.G5.5.5
M.G5.5.10
M.G10.5.5
M.G10.0.10
M.G15.0.5
M.G15.3.10
Diamicron
Figure 4.11: Percentage of the drug dissolved as a function of time (min) within pH calibrated medium form simulating either a
acidic or alkaline gastric environments
85
This release is indicative of a fast release of the drug. Burst release can be advantageous in
certain applications e.g., drugs which require an immediate release and continuous release for
predetermined time interval, though in certain dosage form designs which require a slow initial
release, a burst release could be disadvantageous (Huang & Brazel, 2001:121-135). The burst
release may be attributed to the existence of cavities within the bead structure as was evident
from the SEM micrographs. It was postulated that the existence of these cavities might be
beneficial for liquid penetration into the bead structure and thereby benefited the dissolution rate
of the drug. This can contribute to the burst release seen within the first few minutes of the
dissolution study from this formulation. However, the remaining drug content (± 40 - 50%) was
released over the remaining period of the study. The corresponding release profile seen here
correlates with figure 2.2.
Although this formulation did not mimic the release profile of the control precisely, it is still
evident that extended release was observed over a period of approximately 6 h. Formulation
M.G15.3.10 exhibited a dissolution profile similar to the control, Diamicron®, under the test
conditions which indicated that it was possible to prepare a multiple pellet system that rendered
modified release of gliclazide.
Table 4.5: Mean dissolution time and similarity factor values for each bead formulation and
Diamicron®
Formulation Mean Dissolution Time
(min) Similarity Factor (f2)
M.G5.3.5 307.1 ± 27.17 37.3 ± 3.55
M.G5.5.10 201.7 ± 5.34 45.8 ± 3.11
M.G10.5.5 226.9 ± 9.21 49.9 ± 3.76
M.G10.0.10 268.3 ± 3.53 45.8 ± 27.00
M.G15.0.5 281.1 ± 9.79 42.8 ± 2.63
M.G15.3.10 215.6 ± 6.42 50.6 ± 4.45
C.G5.5.5 120.0 ± 54.45 27.9 ± 5.07
Diamicron® 211.7 ± 13.59
86
In table 4.4, the average MDT-values and similarity factor values of the different formulations
are given. This statistical analysis from the dissolution profile (figure 4.11 and table 4.4)
provided a more empirical analysis regarding the similarity between dissolution profiles of the
respective bead formulations and the control. It also provided data concerning the mean
dissolution time.
From table 4.4 it could be seen that, several of the formulations containing Avicel© had MDT
values notably longer (20 - 45%) than Diamicron®. A longer MDT indicates a slower rate of
release and a low MDT is indicative of a faster release. From the MDT values it could be seen
that M.G5.3.5 depicted a MDT 45.50% higher than Diamicron®. The cassava formulation
(C.G5.5.5) illustrated a dissolution profile, with an MDT of 43.60% less than that of the control.
This low MDT (119.99 ± 54.451) could be attributed to the rapid dissolution of the beads within
the first 15 min of the study. Formulation M.G15.3.10 exhibited a MDT-value similar to that of
Diamicron®. This correlates with the profile seen in figure 4.11; and the f2-value of M.G15.3.10
exceeded 50%, thus rendering M.G15.3.10 similar to the control. Although extended release for
formulation C.G5.5 can be observed from figure 4.11 it exhibited the fastest MDT value
(119.99 ± 54.541 min) and lowest similarity factor value (27.86 ± 5.071); and therefore it can be
concluded that the cassava-containing formula did exhibited a dissolution profile that differed
markedly from the profile observed for the control. Based on the dissolution parameters and
dissolution profiles depicted in figure 4.11, all formulations exhibited extended release although
to different degrees in comparison to the control, Diamicron® tablets. This highlights the
versatility of a multiple unit pellet system in modifying drug release.
HPMC has been described as an aid for modified drug release (Moodley et al., 2011:18-43;
Okunlola, 2015:1). This attribute could be ascribed to the high swellability and hydrophilic
nature of HPMC’s polymer matrix and consequent dissolution of the expanded matrix during
dissolution (Jiyauddin et al., 2014: Moodley et al., 2012:18-43; Oliveira et al., 2013:2; Scholtz
et al.¸ 2014:486-501; Siepmann & Peppas, 2012:163-173; Uhrich et al., 1999:3181-3198).
These various characteristics of HPMC contributed to prolonged MDT-values of the Avicel®
formulations.
4.7 SUMMARY
Moisture content as determined by Karl-Fischer titration indicated the starches were highly
susceptible to humidity. Conversely, powders with lower moisture content proved
87
advantageous in regards to flow. IR-spectroscopy indicated the relationship betweenthe two
starches and FTIR-analysis confirmed the difference between the two starch samples.
The bead formulations were evaluated with regards to surface morphology and internal
structure by means of SEM. SEM-micrographs, revealed that although not perfectly spherical,
beads were successfully prepared. The bead formulation containing cassava exhibited a rough
surface that could be accredited to the morphology of the cassava particles itself. Bead
formulations containing Avicel® demonstrated a more tightly packed internal structure. Size
analysis indicated that the majority of bead samples were larger than 1000 µm. Furthermore,
the starch powders exhibited poor powder flow properties, whereas all of the bead formulations
exhibited good powder flow properties. This was confirmed by all the powder flow parameters
that were determined.
With regards to swelling and erosion studies, the cassava-containing beads formulation
disintegrated quickly and swelling could therefore not be determined. However, Avicel® swelling
for all the Avicel®-containing formulations was evident. Moreover, it appeared that the inclusion
of HPMC resulted in an increased degree of bead swelling; however, its inclusion also resulted
in an increase in bead erosion.
Parameters and data obtained from the dissolution studies provided evidence which promotes
the application of cassava starch as an excipient in the production of modified release SODFs.
Depending on the excipients and manufacturing parameters used in production, the dissolution
behaviour can be similar to that of a commercial product or even be significantly greater.
Chapter 5 will conclude the study with a general summary and conclusion as well as possible
avenues for further study.
88
Chapter 5
GENERAL SUMMARY AND FUTURE PROSPECTS
5.1 SUMMARY & FUTURE PROSPECTS
The aim of this study as stated in Chapter 1, was to investigate the possible application of
cassava starch as a modified release excipient; and whether a modified SODF could be
manufactured. Chapter 2 provided a literature overview regarding SODF manufacturing,
different types of SODFs and excipients used to manufacture SODFs. It continued with the
description of starch as a versatile excipient and the selected starch source, cassava. Cassava,
one of the dominant sources of starch, is widely spread throughout sub-tropic environments and
easily cultivated. Being a biodegradable and renewable source of starch, cassava is promising
in many industries, e.g. clothing, paper, pharmaceutical, etc.. It is rich in amylopectin and
amylose, the two dominant biopolymers found within cassava. These polymers are cross-linked
forming a natural occurring matrix. Polymer-matrices have proven advantageous in the
development of various dosage forms, especially in modified release SODFs. For the above
mentioned reasons, the use of Cassava as a renewable source of starch can be advocated with
merit. Chapter 3 dealt with the experimental methods that were employed to determine the
physical characteristics, size, morphology and flow properties of the starch and extrusion-
spheronised beads.
From the results, it was evident that cassava starch was a hygroscopic starch, with weak flow
properties. This poor flowability would adversely affect the ability of the cassava starch to be
used in terms of direct compression. The moisture content of the powders was determined as
15.82 ± 0.339% and 11.84 ± 0.156% respectively for the donated and purchased starch. IR-
spectra were obtained to provide a fingerprint in order to compare the two starches. Thermo-
graphs were constructed for both starches and this indicated whether the moisture was present
as part of the chemical structure of the powder or whether it was present due to hygroscopicity.
From the thermo-graphs it could be seen that the moisture was present due to hygroscopicity
and not part of the chemical structure. This indicated the possibility of altering the powder flow
by simply heating the powder in a regulated oven.
By manufacturing beads from cassava the flow was drastically improved. The manufacturing of
cassava beads were difficult due to the presence of the hydrophobic drug, gliclazide, which was
89
selected as a model drug due to its importance as a second line treatment in Diabetes Mellitus
Type-2. Several successful bead formulations were manufactured with Avicel® as filler. The
production of Avicel® provided a product to which a bead manufactured frm cassava can be
compared. It was also evident from the study, that HPMC played a vital role in the manufacture
of quality beads. HPMC containing beads depicted a higher quality in terms of spherocity, which
corroborates findings by Dukiƈ-Ott et al. (2009:42-43) and Ghandi et al., (1999:166-168).
HPMC furthermore contributed to the modification of drug release (Moodley et al., 2012:21-36;
Oliveira et al., 2013:2). Multi-unit particulate system tablets could not be manufactured with
cassava beads due to the inability of the tablet to hold its shape. These tablets crumbled into
deformed beads. It was opted to encapsulate different intact bead samples in hard gelatine
capsules.
SEM-micrographs provided visual data relating to the shape of the powder particles, as well as
the whole beads, their internal structure and external morphology. These micrographs indicated
that the powder particles were spherical and exhibited surface irregularities. Size-distribution
analyses on the cassava powder particles and bead formulations were conducted. The D(0.9)
value for the starch particles were < 100 µm and all bead samples were < 1000 µm. Flow
properties of the powders and beads were characterised using various parameters (e.g. critical
orifice diameter, angle of repose, compressibility, etc.). Collectively, these parameters indicated
that the starches exhibited poor flow and that the beads portrayed acceptable flow. Friability
analyses illustrated that that the cassava bead formulations might not have acceptable physical
stability to withstand transport or handling.
Dissolution studies were conducted over a 12 h period. Samples were taken at predetermined,
time intervals and analysed with a UV-spectrometer for gliclazide content. Dissolution profiles
were characterised by means of the mean dissolution time (MDT) and similarity factor (f2).
Cassava starch beads provided modified drug release, where 60% of the total drug content was
pharmaceutically available within the first 15 min of the study. The remaining 40% dissolved
slowly over the remaining duration of the study. These beads depicted a MDT markedlyf less
than that of Diamicron®, at approximately 120.00 ± 13.59 min compared to a MDT value of
211.70 ± 13.59 min for the Diamicron® tablets. The release profile of the Cassava beads were
not similar to the control (Diamicron®) as evidenced by a similarity factor value of
27.86 ± 5.07%. Avicel® containing formulations exhibited MDT values ranging from
201.68 ± 5.34 to 307.05 ± 27.17 min. HPMC did not appear to affect drug release as no clear
tendency could be identified related to HPMC content.
90
5.2 FUTURE PROSPECTS
This study employed cassava starch, a sustainable, renewable and cost-effective, source of
starch as an excipient in modified release SODFs. Test formulations contained a binder, HPMC
and the poor water soluble drug, gliclazide. Extrusion-spheronisation was the chosen method of
bead manufacturing for the production of a modified release SODF. From the results of this
study, the following prospects for future studies are identified:
1) The effects of a water soluble drug can be investigated, instead of a poorly water soluble
drug, to evaluate the possible manufacturing of beads with cassava starch.
2) Multi-unit particulate system MUPS tablets containing cassava starch should be re-
manufactured, by altering the method of bead drying. Instead of using a regulated oven,
the beads could be lyophilised. This might provide a less rigid bead, capable of deforming
during compression and improving cohesion, which in turn could provide a more stable and
resilient MUPS tablet.
3) Another consideration in the manufacture of MUPS tablets from beads in this study, is the
mixing of a binder or filler to provide cohesion between the individual beads, before
compression.
4) Investigation of cassava starch in combination with other fillers, e.g. Avicel®, Microcelac®,
etc., in order to explore cassava starch as a release modifying excipient in bead
formulations and to evaluate the effect of these additions on bead manufacture.
5) An enteric coating can be applied to the cassava starch beads to prevent fast/immediate
dissolution of the cassava starch in the acidic environment of the stomach, which in turn
can influence the release profile of bead formulations containing this starch.
91
REFERENCES
I hereby confirm and declare that this study is my own and where
suitable reference and acknowledgement is given to external parties
and sources
~||~
ABDUL, S. & PODDAR, S. 2004. A flexible technology for modified release of drugs: multi
layered tablets. Journal of controlled release, 97:393-405.
ADDO, J., SMEETH, L. & LEON, D.A. 2007. Hypertension in Sub-Saharan Africa a systematic
review. Hypertension, 50:1012-1018.
AKHGARI, A., ABBASPOUR, M., REZAEE, S. & KUCHAK, A. 2007. Evaluation of the Swelling,
Erosion and Drug Release from Polysaccharide Matrix Tablets based on Pectin and Inulin.
Jundushapur journal of natural pharmaceutical products, 6:51-58.
ALDERBORN, G. 2002. Tablets and compaction. (In Aulton, M.E. ed. Pharmaceutics: the
science of dosage form design. 2nd ed. London: Churchill Livingstone. P. 397-440)
ALDERBORN, G. 2007. Tablets and compaction. (In Aulton, M.E. ed. Pharmaceutics: the
science of dosage form design. 3nd ed. London: Churchill Livingstone. P. 504-549)
ALLEN, L.V., Popovich, N.G., Ansel, H.C. 2011. Ansel’s Pharmaceutical dosage forms and drug
delivery systems. 9th ed. Philadelphia. Wolters Kluwer Health: Lippincott, Williams & Wilkins.
ASHFORD, M. 2007. Bioavailability – physicochemical and dosage form factors (In Aulton, M.E.
ed Pharmaceutics: the science of dosage form design. 3rd ed. Edinburgh: Churchill Livingstone).
AUCAMP, M., STIEGER, N., BARNARD, N. & LIEBENBERG, W. 2013. Solution-mediated
phase transformation of different roxithromycin solid-state forms: Implications on dissolution and
solubility. International journal of pharmaceutics, 449:18-27.
AULTON, M.E. & WELLS, T. 2002. Pharmaceutics: The science of dosage form design,
London: Churchill Livingstone.
Aulton, M.E., & Taylor, K.M. 2013. Aulton's pharmaceutics: the design and manufacture of
medicines. Elsevier Health Sciences
92
BADAWI, A.A. 2004. The Social Dimension of Globalization and Health. Perspectives on global
development and technology, 3:73-90.
BARDONNET, P., FAIVRE, V., PUGH, W., PIFFARETTI, J. & FALSON, F. 2006.
Gastroretentive dosage forms: Overview and special case of Helicobacter pylori. Journal of
controlled release, 111:1-18.
BARTELS, D. 2004. Adherence to oral therapy for type 2 diabetes: opportunities for enhancing
glycemic control. Journal of the american academy of nurse practitioners, 16:8-16
BAYOR, M. T., TUFFOUR, E. & LAMBON, P. S. 2013. Evaluation of Starch from New Sweet
Potato Genotypes for use as A Pharmaceutical Diluent, Binder or Disintegrant. Journal of
applied pharmaceutical science, 3:17-23
BENEKE, C. E., VILJOEN, A. M. & HAMMAN, J. H. 2009. Polymeric plant-derived excipients in
drug delivery. Molecules, 14:2602-2620.
BHASKARAN, S. & LAKSHMI, P. 2010. Extrusion spheronization—A review. International
journal of pharmeutical technology research,2:2429-2433.
BRITISH PHARMACOPOEIA. 2015. https://www.pharmacopoeia.com/BP2015
Date of access: 30 October 2015
BUDIASIH, S., JIYAUDDIN, K., LOGAVINOD, N., KALEEMULLAH, M., JAWAD, A., SAMER,
A., FADLI, A. & EDDY, Y. Optimization of Polymer Concentration for Designing of Oral Matrix
Controlled Release Dosage Form. UK journal of pharmaceutical and biosciences, 2:54-61
BÜHLER, V. 2003. Kollidon® Polyvinylpyrrolidone for the pharmaceutical industry 7th ed.
Ludwigshaven: BASF
BUYS, G.M. 2005. Formulation of a chitosan multi-dosage form for drug delivery to the colon.
Potchefstroom North-West University (Thesis-PhD).
CASAS, M., FERRERO, C. & JIMÉNEZ-CASTELLANOS, M.R. 2010. Graft tapioca starch
copolymers as novel excipients for controlled-release matrix tablets. CarbohydratepPolymers,
80:71-77.
CASSAVA IMAGE https://theglyptodon.files.wordpress.com/2011/06/cassava.jpg Date of
access: 30 March 2014
93
CHARLES, A.L., CHANG, Y.H., KO, W.C., SRIROTH, K. & HUANG, T.C. 2005. Influence of
amylopectin structure and amylose content on the gelling properties of five cultivars of cassava
starches. Journal of agricultural and food chemistry, 53:2717-2725.
CHEBOYINA, S. & O'HAVER, J.H. 2004. Process and apparatus for producing spherical pellets
using molten solid matrices. Google Patents.
CHINYEMBA, P. 2012. Use of Aloe vera and Aloe marlothii materials as excipients in beads
produced by extrusion-spheronization. Potchefstroom North-West University (Dissertation –
MSc).
CHITEDZE, J., MONJEREZI, M., SAKA, J. & STEENKAMP, J. 2012. Binding Effect of Cassava
Starches on the Compression and Mechanical Properties of Ibuprofen Tablets. Journal of
applied pharmaceutical sciences, 4:31-37
COLOMBO, P., BETTINI, R., SANTI, P. & PEPPAS, N.A. 2000. Swellable matrices for
controlled drug delivery: gel-layer behaviour, mechanisms and optimal performance.
Pharmaceutical science and technology today, 3:198-204.
CHOPRA, S., VENKATESAN, N. & BETAGRI, G.V. 2013 Formulation of lipid bearing pellets
as a delivery system for poorly soluble drugs. International journal of pharmaceutics,
446:136-144
CORDAIN, L., EATON, S.B., SEBASTIAN, A., MANN, N., LINDEBERG, S., WATKINS, B.A.,
O’KEEFE, J.H. & BRAND-MILLER, J. 2005. Origins and evolution of the Western diet: health
implications for the 21st century. The American journal of clinical nutrition, 81:341-354.
COSTA, P. & LOBO, J. M. S. 2001. Modeling and comparison of dissolution profiles. European
journal of pharmaceutical sciences, 13:123-133.
CROUTER, A. & BRIENS, L. 2014. The effect of moisture on the flowability of pharmaceutical
excipients. American association of pharmaceutical scientist PharmSciTech, 15:65-74.
DALL, T.M., ZHANG, Y., CHEN, Y.J., QUICK, W.W., YANG, W.G. & FOGLI, J. 2010. The
economic burden of diabetes. Health affairs, 29:297-303.
DAS, N.G. & DAS, S.K. 2003. Controlled release of oral dosage forms. Pharmaceutical
technology, 15:10-17.
94
DAY, C. & BAILEY, C.J. 2011. Obesity in the pathogenesis of type 2 diabetes. The british
journal of diabetes and vascular disease, 11:55-61.
DE KOCK, J.M. 2005. Chitosan as a multipurpose excipient in directly compressed minitablets.
North-West University (Thesis – PhD).
DE FLOOR, I., DEHING, I. & DELCOUR, J. 1998. Physico‐Chemical Properties of Cassava
Starch. Starch‐Stärke, 50:58-64.
DELAMATER, A.M. 2006. Improving patient adherence. Clinical diabetes, 24:71-77.
DEY, N.C., MAJUMDAR, S. & RAO, M.E.B. 2008 Multiparticulate Drug Delivery Systems for
Controlled Release. Tropical journal of pharmaceutical research, 7:1067-1075
DHANDAPANI, N.V. 2012. Pelletization by Extrusion-Spheronization-A detailed review. The all
results journals: Biology, 3:10-23.
DIABETES http://www.who.int/diabetes/facts/en/ Date of access: 25 August 2013
DRESSER, G.K., SPENCE, J.D. & BAILEY, D.G. 2000. Pharmacokinetic-pharmacodynamic
consequences and clinical relevance of cytochrome P450 3A4 inhibition. Clinical
pharmacokinetics, 38:41-57.
DRESSMAN, J.B., AMIDON, G L., REPPAS, C. & SHAH, V.P. 1998. Dissolution testing as a
prognostic tool for oral drug absorption: immediate release dosage forms. Pharmaceutical
research, 15:11-22.
DUKIĆ-OTT, A., THOMMES, M., REMON, J.P., KLEINEBUDDE, P. & VERVAET, C. 2009.
Production of pellets via extrusion–spheronisation without the incorporation of microcrystalline
cellulose: a critical review. European journal of pharmaceutics and biopharmaceutics, 71:38-46.
DUMOULIN, Y., CARRIERE, F. & INGENITO, A. 1998. Manufacture of cross-linked amylose
useful as an excipient for control release of active compounds. Google Patents.
DURAIAPPAH, A. K. 1998. Poverty and environmental degradation: a review and analysis of
the nexus. World development, 26:2169-2179.
EMERY, E., OLIVER, J., PUGSLEY, T., SHARMA, J. & ZHOU, J. 2009. Flowability of moist
pharmaceutical powders. Powder technology, 189:409-415.
95
FALDU, B. & ZALAVADIYA, B. 2012. LUBRICANTS: FUNDAMENTALS OF TABLET
MANUFACTURING. International journal of research in pharmacy and chemistry, 2:2231-
2781.FAMÁ, L., FLORES, S.K., GERSCHENSON, L. & GOYANES, S. 2006. Physical
characterization of cassava starch biofilms with special reference to dynamic mechanical
properties at low temperatures. Carbohydrate polymers, 66:8-15.
FAMÁ, L., GOYANES, S. & GERSCHENSON, L. 2007. Influence of storage time at room
temperature on the physicochemical properties of cassava starch films. Carbohydrate olymers,
70:265-273.
FOWLER, M.J. 2007. Diabetes treatment, part 2: oral agents for glycemic management. Clinical
diabetes, 25:131-134.
FRIZON, F., DE OLIVEIRA ELOY, J., DONADUZZI, C.M., MITSUI, M.L. & MARCHETTI, J.M.
2013. Dissolution rate enhancement of loratadine in polyvinylpyrrolidone K-30 solid dispersions
by solvent methods. Powder technology, 235:532-539.
Gandhi, B., & Baheti, J. 2013. Multiparticulates drug delivery systems: a review. Indian journal
of pharmaceutical chemical sciences, 2:1620-6.
GANDHI, R., KAUL, C. L. & PANCHAGNULA, R. 1999. Extrusion and spheronization in the
development of oral controlled-release dosage forms. Pharmaceutical science and technology
today, 2:160-170.
GARR, J. & RUBINSTEIN, M. 1992. The influence of moisture content on the consolidation and
compaction properties of paracetamol. International journal of pharmaceutics, 81:187-192.
GELDART, D., ABDULLAH, E., HASSANPOUR, A., NWOKE, L. & WOUTERS, I. 2006.
Characterization of powder flowability using measurement of angle of repose. China
particuology, 4:104-107.
GHORI, M. U., GINTING, G., SMITH, A.M. & CONWAY, B.R. 2014. Simultaneous quantification
of drug release and erosion from hypromellose hydrophilic matrices. International journal of
pharmaceutics, 465:405-412.
GOYAL, A., SHUKLA, P. & SRIVASTAVA, A. 2009. Factors influencing drug release
characteristic from hydrophilic polymer matrix tablet. Asian journal of pharmcological clinic
research, 2:93-98.
96
GUPTA, B.P., THAKUR, N., JAIN, N.P., BANWEER, J. & JAIN, S. 2010. Osmotically controlled
drug delivery system with associated drugs. Journal of pharmacy and pharmaceutical sciences,
13:571-588.
HALL, V., THOMSEN, R.W., HENRIKSEN, O. & LOHSE, N. 2011. Diabetes in Sub Saharan
Africa 1999-2011: epidemiology and public health implications. A systematic review. BMC public
health, 11:564.
HANCOCK, B.C., VUKOVINSKY, K.E., BROLLEY, B., GRIMSEY, I., HEDDEN, D., OLSOFSKY,
A. & DOHERTY, R. A. 2004. Development of a robust procedure for assessing powder flow
using a commercial avalanche testing instrument. Journal of pharmaceutical and biomedical
analysis, 35:979-990.
HART, A. 2015. Effect of Particle Size on Detergent Powders Flowability and Tabletability.
Journal of chemical engineering and process technology, 6:1.
HIRANI, J.J., RATHOD, D.A. & VADALIA, K.R. 2009. Orally disintegrating tablets: a review.
Tropical journal of pharmaceutical research, 8.
HORN, E. 2008. Development of a composite index for pharmaceutical powders. Potchefstroom
North-West University (Dissertation-MSc)
HUANG, X. & BRAZEL, C.S. 2001. On the importance and mechanisms of burst release in
matrix-controlled drug delivery systems. Journal of controlled release, 73:121-136.
HUANG, Z.Q., LU, J.P., LI, X.H. & TONG, Z.F. 2007. Effect of mechanical activation on physico-
chemical properties and structure of cassava starch. Carbohydrate Polymers, 68:128-135.
IBRAGIMOVA, M. M. & IKRAMOV, L.T. 2015. Analysis of Glimepiride in Human Blood and
Urine by Thin-Layer Chromatography and UV-Spectrophotometry. The bulletin of legal
medicine, 20:71-75.
INGLE, L.M., WANKHADE, V.P., UDASI, T.A. & TAPAR, K.K. 2013. New Approaches for
Development and Characterization of SMEDDS. International journal of pharmacy and
pharmaceutical science research, 3:7-14.
ISPAS-SZABO, P., RAVENELLE, F., HASSAN, I., PREDA, M. & MATEESCU, M.A. 1999.
Structure–properties relationship in cross-linked high-amylose starch for use in controlled drug
release. Carbohydrate research, 323:163-175.
97
JALLO, L.J., GHOROI, C., GURUMURTHY, L., PATEL, U. & DAVÉ, R.N. 2012. Improvement of
flow and bulk density of pharmaceutical powders using surface modification. International
journal of pharmaceutics, 423:213-225.
JAMADAR, S.A., MULYE, S.P., KAREKAR, P.S., PORE, Y.V. & BURADE, K.B. 2011. Development and
validation of UV spectrophotometric method for the determination of Gliclazide in tablet dosage form.
Der pharma chemical, 3:338-343
JAVADZADEH, Y., VAZIFEHASL, Z., DIZAJ, S.M. & MOKHTARPOUR, M. 2015. Spherical
Crystallization of Drugs. Advanced topics in crystallization, Chapter 4:85-104.
JEWESSON, P.J. 1996. Economic impact of intravenous-to-oral antibacterial stepdown therapy.
Clinical drug investigation, 11:1-9.
JIVRAJ, M., MARTINI, L.G. & THOMSON, C. M. 2000. An overview of the different excipients
useful for the direct compression of tablets. Pharmaceutical science andtechnology today, 3:58-
63.
JIYAUDDIN, K., SUNG, K., SAMER, A., KALEEMULLAH, M., RASHA, S., BUDIASIH, S.,
JAWAD, A., RASNY, R., GAMAL, E. & JUNAINAH, H. 2014. Comparative study on the effect of
hydrophilic and hydrophobic polymers on the dissolution rate of a poorly water soluble drug. UK
journal of pharmaceutical and biosciences, 2:51-61
JOSHI, A.K., PUND, S.V., NIVSARKAR, M.A., VASU, K.K. & SHISHOO, C.J. 2011. Exploring
the potential of polacrilin potassium as a novel super disintegrant in microcrystalline cellulose
based pellets prepared by extrusion-spheronization. Chronicles of young scientists, 2:111.
KALRA, S. & GUPTA, Y. 2015. Sulfonylureas. The journal of the pakistan medical association,
65:101-104.
KALRA, S., MADHU, S. & BAJAJ, S. 2015. Sulfonylureas: Assets in the past, present and
future. Indian journal of endocrinology and metabolism, 19:314.
KARDAS, P. 2005. The DIACOM study (effect of Dosing frequency of oral Antidiabetic agents
on the Compliance and biochemical control of type 2 diabetes). Diabetes, Obesity and
Metabolism, 7:722-728.
98
KASSAB, N.M., AMARAL, M.S.D., SINGH, A.K. & SANTORO, M.I.R.M. 2010. Development and
validation of UV spectrophotometric method for determination of levofloxacin in pharmaceutical
dosage forms. Química Nova, 33:968-971.
KATZUNG, B., MASTERS, S. & TREVOR, A. 2009. Basic and Clinical Pharmacology. McGraw-
Hill Companies. Inc., New York.
KAWACHI, I., KENNEDY, B.P., LOCHNER, K. & PROTHROW-STITH, D. 1997. Social capital,
income inequality, and mortality. American journal of public health, 87:1491-1498.
KHAN, A., MALVIYA, R. & SHARMA, P.K. 2014. Multi Unit Drug Delivery System–A Brief
Review of Pelletization Technique. World applied sciences journal, 31:2137-2140.
KHAN, G.M. 2001. Controlled release oral dosage forms: Some recent advances in matrix type
drug delivery systems. The sciences, 1:350-354.
KIM, E. H.J., CHEN, X.D. & PEARCE, D. 2005. Effect of surface composition on the flowability
of industrial spray-dried dairy powders. Colloids and surfaces b: biointerfaces, 46:182-187.
KO, K.H., RAWAL, A. & SAHAJWALLA, V. 2014. Analysis of thermal degradation kinetics and
carbon structure changes of co-pyrolysis between macadamia nut shell and PET using
thermogravimetric analysis and 13 C solid state nuclear magnetic resonance. Energy
conversion and management, 86:154-164.
KOESTER, M. AND THOMMES, M. 2010. New insights into the pelletization mechanism by extrusion/epheronization. American association of pharmaceutical scientist PharmSciTech, 11:1549-1551.
KORHONEN, O., RAATIKAINEN, P., HARJUNEN, P., NAKARI, J., SUIHKO, E., PELTONEN,
S., VIDGREN, M. & PARONEN, P. 2000. Starch acetates—multifunctional direct compression
excipients. Pharmaceutical research, 17:1138-1143.
KRAJACIC, A. & TUCKER, I.G. 2003. Matrix formation in sustained release tablets: possible
mechanism of dose dumping. International journal of pharmaceutics, 251:67-78.
KRULL, E.S., MCBEATH, A.V., SMERNIK, R.J. AND LEHMANN, J. 2014. The influence of feedstock and production temperature on biochar carbon chemistry: A solid-state 13C NMR study. Biomass and bioenergy, 60:121-129
99
KUMAR, S., DAS, B. & RAJU, S.R. 2012. Formulation and Evaluation of Multiunit Pellet System
of Venlafaxine Hydrochloride. Journal of pharmaceutical and biomedical sciences, 18:182-3.
LAHIRI, S.W. 2012. Management of type 2 diabetes: what is the next step after metformin?
Clinical diabetes, 30:72-75.
LAJOINIE, A., HENIN, E., KASSAI, B. & TERRY, D. 2014. Solid oral forms availability in
children: A cost saving investigation. British journal of clinical pharmacology, 78:1080-1089.
LAMBRECHTS, J.J. 2008. Investigation into the influence of different Kollidon polymers on the
properties of powder mixtures intended for tableting. Potchefstroom North-West University
(Dissertation – MSc).
LANDILLON, V., CASSAN, D., MOREL, M.H. & CUQ, B. 2008. Flowability, cohesive, and
granulation properties of wheat powders. Journal of food engineering, 86:178-193.
LAVANYA, K., SENTHIL, V. & RATHI, V. 2011. Pelletization technology: a quick review.
International journal of pharmaceutical scientific research, 2:1337-1355.
LAVOIE, F., CARTILIER, L. & THIBERT, R. 2002. New methods characterizing avalanche
behavior to determine powder flow. Pharmaceutical research, 19:887-893.
LAZAR, M. A. 2005. How obesity causes diabetes: not a tall tale. Science, 307:373-375.
LEBOVITZ, H. E. 1999. Type 2 diabetes: an overview. Clinical chemistry, 45:1339-1345.
LEMIEUX, M., GOSSELIN, P. & MATEESCU, M.A. 2009. Carboxymethyl high amylose starch
as excipient for controlled drug release: Mechanistic study and the influence of degree of
substitution. International journal of pharmaceutics, 382:172-182.
LEMMER, H., STIEGER, N., LIEBENBERG, W. & CAIRA, M.R. 2012. Solvatomorphism of the
antibacterial dapsone: x-ray structures and thermal desolvation kinetics. Crystal growth &
design, 12:1683-1692.
LENAERTS, V., DUMOULIN, Y. & MATEESCU, M.A. 1991. Controlled release of theophylline
from cross-linked amylose tablets. Journal of controlled release, 15:39-46.
LENAERTS, V., MOUSSA, I., DUMOULIN, Y., MEBSOUT, F., CHOUINARD, F., SZABO, P.,
MATEESCU, M., CARTILIER, L. & MARCHESSAULT, R. 1998. Cross-linked high amylose
100
starch for controlled release of drugs: recent advances. Journal of controlled release, 53:225-
234.
LIU, L. X., MARZIANO, I., BENTHAM, A., LITSTER, J.D., WHITE, E. & HOWES, T. 2008. Effect
of particle properties on the flowability of ibuprofen powders. International journal of
pharmaceutics, 362:109-117.
LOURENÇO, F.R., GHISLENI, D.D.M., YAMAMOTO, R.N. & PINTO, T.D.J.A. 2013.
Comparison of dissolution profile of extended-release oral dosage forms-two one-sided
equivalence test. Brazilian journal of pharmaceutical sciences, 49:367-371.
MALLIPEDDI, R., SARIPELLA, K.K. & NEAU, S.H. 2010. Use of coarse ethylcellulose and PEO
in beads produced by extrusion–spheronization. International journal of pharmaceutics, 385:53-
65.
MALLIPEDDI, R., SARIPELLA, K. K. & NEAU, S. H. 2014. Use of fine particle ethylcellulose as
the diluent in the production of pellets by extrusion-spheronization. Saudi pharmaceutical
journal, 22:360-372.
MANDAL, S., BASU, S.K. & SA, B. 2009. Sustained release of a water-soluble drug from
alginate matrix tablets prepared by wet granulation method. American association of
pharmaceutical scientists PharmSciTech, 10:1348-1356.
MARAIS, E.B. 2013 Permeation of excised intestinal tissue by insulin released from Eudragit®
L100/Trimethyl chitosan chloride microspheres. Potchefstroom North-West University
(Dissertation-MSc).
Martin AN. Physical Pharmacy: Physical Chemical Principles in the Pharmaceutical Sciences,
4th edition, Lippincott Williams & Wilkins: USA, 1993.
MCCULLOCH, D.K., MUNSHI, M., NATHAN, D. M., SCHMADER, K. E. & MULDER, J. E.
Treatment of type 2 diabetes mellitus in the older patient.
MEYROWITSCH, D.W. & BYGBJERG, C. 2007. Global burden of disease-a race against time.
Danish medical bulletin, 54:32-4.
MILLILI, G. & SCHWARTZ, J. 1990. The strength of microcrystalline cellulose pellets: the effect
of granulating with water/ethanol mixtures. Drug development and industrial pharmacy,
16:1411-1426.
101
Molecular and macroscopic structure of amylose and amylopectin.
http://voer.edu.vn/m/carbohydrates/70e79f40 Date of access: 30 March 2014
MOODLEY, K., PILLAY, V., CHOONARA, Y. E., DU TOIT, L C., NDESENDO, V.M., KUMAR,
P., COOPPAN, S. & BAWA, P. 2011. Oral drug delivery systems comprising altered geometric
configurations for controlled drug delivery. International journal of molecular sciences, 13:18-43.
MOORE, J.W. & FLANNER, H.H. 1996. Mathematical comparison of dissolution profiles.
Pharmaceutical technology, 20, 64-74.
MOORTHY, S.N. 2002. Physicochemical and functional properties of tropical tuber starches: a
review. Starch‐Stärke, 54:559-592.
MOTALA, A. & RAMAIYA, K. 2010. Diabetes: the hidden pandemic and its impact on Sub-
saharan Africa. Diabetes leadership forum
MULLARNEY, M.P., BEACH, L.E., DAVÉ, R.N., LANGDON, B.A., POLIZZI, M. &
BLACKWOOD, D.O. 2011. Applying dry powder coatings to pharmaceutical powders using a
co-mill for improving powder flow and bulk density. Powder tTechnology, 212:397-402.
MULTIPARTICULATE DRUG DELIVERY SYSTEMS.
http://www.slideshare.net/bknanjwade/multiparticulate-drug-delivery-systems Date of access:
25 April 2014
MUSTAFA, M.E., NUR, A.O., OSMAN, Z.A. & AHMED, S.A. 2014. Influence of drug solubility
and polymers supply source on the physical performance of matrix tablets. International journal
of pharmacy and pharmaceutical sciences, 6:301-312
NEWTON, J., CHAPMAN, S. & ROWE, R. 1995. The influence of process variables on the
preparation and properties of spherical granules by the process of extrusion and spheronisation.
International journal of pharmaceutics, 120:101-109.
NOKHODCHI, A. 2005. Effect of moisture on compaction and compression. Pharmaceutical
Technology, 646-66.
NUWAMANYA, E., BAGUMA, Y., EMMAMBUX, N., TAYLOR, J. & PATRICK, R. 2010.
Physicochemical and functional characteristics of cassava starch in Ugandan varieties and their
progenies. Journal of plant breeding and crop science, 2:001-011.
102
OKUNLOLA, A., ODEKU, O.A. & PATEL, R. P. 2012. Formulation optimization of floating
microbeads containing modified Chinese yam starch using factorial design. Journal of excipients
and food chemicals, 3:17-25.
OLIVEIRA, P.R., MENDES, C., KLEIN, L., SANGOI, M.D.S., BERNARDI, L. S. & SILVA, M.A.
S. 2013. Formulation development and stability studies of norfloxacin extended-release matrix
tablets. BioMed research international, 2013:1-9
Oxford dictionary definition of morphology.
http://www.oxforddictionaries.com/definition/english/morphology Date of access: 20 March
2014.
PAINE, M F., HART, H.L., LUDINGTON, S.S., HAINING, R.L., RETTIE, A.E. & ZELDIN, D.C.
2006. The human intestinal cytochrome P450 “pie”. Drug metabolism and disposition, 34:880-
886.
PANTEN, U., SCHWANSTECHER, M. AND SCHWANSTECHER, C. 1996. Sulfonylurea
receptors and mechanism of sulfonylurea action. Experimental and clinical endocrinology
and diabetes, 104:1-9
PATEL, A., RAY, S. & THAKUR, R.S. 2006. Invitro evaluation and optimization of controlled
release floating drug delivery system of metformin hydrochloride. DARU Journal of
pharmaceutical sciences, 14:57-64.
PATEL, P.S., RAVAL, J. P. & PATEL, H.V. 2010. Review on the pharmaceutical applications of
hot melt extruder. Asian journal of pharmaceutical and clinical research, 3.
PERIOLI, L., D’ALBA, G. & PAGANO, C. 2012. New oral solid dosage form for furosemide oral
administration. European journal of pharmaceutics and biopharmaceutics, 80:621-629.
Physicochemical properties of gliclazide.
http://www.chemicalbook.com/ChemicalProductProperty_EN_CB5113462.htm Date of access:
30 May 2014
Physicochemical properties of gliclazide. http://www.drugbank.ca/drugs/DB01120 Date of
access: 30 May 2014
Radial extruder. http://spheronizer.com/html/extrusion.html Date of access: 20 April 2014
103
REDDY, S.G. 2006. Globalisation, labour markets, and social outcomes in developing countries.
Available at SSRN 942087.
Reddy, R.B., Malleswari, K., Prasad, G., & Pavani, G. 2012. Colon targeted drug delivery
system: a review. International journal of pharmaceutical science and research, 4:42-54.
REMKO, M. 2009. Theoretical study of molecular structure, pKa, lipophilicity, solubility,
absorption, and polar surface area of some hypoglycemic agents. Journal of molecularstructure:
THEOCHEM, 897:73-82.
RENDELL, M. 2004. The role of sulphonylureas in the management of type 2 diabetes mellitus.
Drugs, 64:1339-1358.
REPPAS, C. & NICOLAIDES, E. 2000. Analysis of drug dissolution data. Drugs and the
pharmaceutical sciences, 106:229-254.
ROLLAND-SABATÉ, A., SÁNCHEZ, T., BULÉON, A., COLONNA, P., JAILLAIS, B.,
CEBALLOS, H. & DUFOUR, D. 2012. Structural characterization of novel cassava starches with
low and high-amylose contents in comparison with other commercial sources. Food
hydrocolloids, 27:161-174.
ROY, S., RIGA, A. & ALEXANDER, K. 2002. Experimental design aids the development of a
differential scanning calorimetry standard test procedure for pharmaceuticals. Thermochimica
acta, 392:399-404.
SAHOO, S., PARVEEN, S. & PANDA, J. 2007. The present and future of nanotechnology in
human health care. Nanomedicine: Nanotechnology, Biology and medicine, 3:20-31.
SASTRY, S.V., NYSHADHAM, J.R. & FIX, J.A. 2000. Recent technological advances in oral
drug delivery–a review. Pharmaceutical science and technology today, 3:138-145.
Save and Grow Cassava: A guide to sustainable production 2013.
http://www.fao.org/docrep/018/i3278e/i3278e.pdf Date of access: 30 August 2013
SCHIELE, J.T., QUINZLER, R., KLIMM, H.D., PRUSZYDLO, M.G. & HAEFELI, W.E. 2013.
Difficulties swallowing solid oral dosage forms in a general practice population: prevalence,
causes, and relationship to dosage forms. European journal of clinical pharmacology, 69:937-
948.
104
SCHOLTZ, J., VAN DER COLFF, J., STEENEKAMP, J., STIEGER, N. & HAMMAN, J. 2014.
More good news about polymeric plant-and algae-derived biomaterials in drug delivery systems.
Current drug targets, 15:486-501.
SHAH, A. & MLODOZENIEC, A. 1977. Mechanism of surface lubrication: Influence of duration
of lubricant‐excipient mixing on processing characteristics of powders and properties of
compressed tablets. Journal of pharmaceutical sciences, 66:1377-1382.
SIEGEL, R.A. & RATHBONE, M. J. 2012. Overview of controlled release mechanisms.
Fundamentals and applications of controlled release drug delivery. Springer.
SIEPMANN, J. & PEPPAS, N. 2012. Modeling of drug release from delivery systems based on
hydroxypropyl methylcellulose (HPMC). Advanced drug delivery reviews, 64:163-174.
SIEWERT, M., DRESSMAN, J., BROWN, C. K., SHAH, V.P., AIACHE, J.M., AOYAGI, N.,
BASHAW, D., BROWN, C., BROWN, W. & BURGESS, D. 2003. FIP/AAPS guidelines to
dissolution/in vitro release testing of novel/special dosage forms. American association of
pharmaceutical scientists PharmSciTech, 4:43-52.
SINGH, K., KUMAR, A., LANGYAN, N. & AHUJA, M. 2009. Evaluation of Mimosa pudica seed
mucilage as sustained-release excipient. American association of pharmaceutical scientists
PharmSciTech, 10:1121-1127.
SONNEKUS, J. 2008. Characterization of the flow and compression properties of chitosan.
Potchefstroom North-West University (Dissertation-MSc).
Social determinants of health 2007
http://www.wpro.who.int/health_research/documents/dhs_hr_health_in_asia_and_the_pacific_0
7_chapter_2_social_determinants_of_health.pdf?ua=1 Date of access: 20 August 2013
STANIFORTH, J.N. 2002. Powders comprising anti-adherant materials for use in Dry Powder
Inhalers. Google Patents.
STANIFORTH, J.N. & AULTON, M.E. 2007. Particle size analysis (In Aulton, M.E. ed
Pharmaceutics: the science of dosage form design. 3rd ed. Edinburgh: Churchill Livingstone).
STORPER, M. 2000. Lived effects of the contemporary economy: globalization, inequality, and
consumer society. Public culture:12, 375-409.
105
SUMMERS, M.P & AULTON, M.E. 2007. Granulation (In Aulton, M.E. ed Pharmaceutics: the
science of dosage form design. 3rd ed. Edinburgh: Churchill Livingstone).
Sungthongieen, S., Sriamornsak, P., Pitaksuteepong, T., Somsiri, A., & Puttipipatkhachorn, S.
2004. Effect of degree of esterification of pectin and calcium amount on drug release from
pectin-based matrix tablets. American association of pharmaceutical scientist PharmSciTech, 5,
50–57.
TAKSANDE, A., TAKSANDE, B., KUMAR, A. & VILHEKAR, K. 2008. Malnutrition related
diabetes mellitus. The journal of Mahatma Gandhi institute. 13:19-24
TEUNOU, E. & FITZPATRICK, J. 1999. Effect of relative humidity and temperature on food
powder flowability. Journal of food engineering, 42:109-116.
The great exchange. 2008. http://www.vos.noaa.gov/MWL/dec_08/great_exchange.shtml Date
of access: 30 August 2013
TONUKARI, N.J. 2004. Cassava and the future of starch. Electronic journal of biotechnology,
7:5-8.
TOUSEY, M.D. 2002. The granulation process 101. Pharmaceutical technology, 8-13.
Traina, K., CLOOTS, R., BONTEMPI, S., LUMAY, G., VANDEWALLE, N. AND BOSCHINI, F.
2013. Flow abilities of powders and granular materials evidenced from dynamical tap density
measurement. Powder technology, 235:842-852.
UHRICH, K.E., CANNIZZARO, S.M., LANGER, R.S. & SHAKESHEFF, K.M. 1999. Polymeric
systems for controlled drug release. Chemical reviews, 99:3181-3198.
United States Pharmacopoiea. www.usp.org Date of access 2014 & 2015
VANDERPOEL, D.R., HUSSEIN, M.A., WATSON-HEIDARI, T. & PERRY, A. 2004. Adherence
to a fixed-dose combination of rosiglitazone maleate/metformin hydrochloride in subjects with
type 2 diabetes mellitus: a retrospective database analysis. Clinical therapeutics:26, 2066-2075.
VELASCO, M., MUNOZ-RUIZ, A., MONEDERO, M. & JIMÉNEZ-CASTELLANOS, M. 1995.
Study of flowability of powders. effect of the addition of lubricants. Drug development and
industrial pharmacy, 21:2385-2391.
106
VERVAET, C., BAERT, L. & REMON, J. P. 1995. Extrusion-spheronisation A literature review.
International journal of pharmaceutics, 116:131-146.
VICENTINI, N., DUPUY, N., LEITZELMAN, M., CEREDA, M. & SOBRAL, P. 2005. Prediction of
cassava starch edible film properties by chemometric analysis of infrared spectra. Spectroscopy
letters, 38:749-767.
VILJOEN, J.M., STEENEKAMP, J.H., MARAIS, A.F. & KOTZÉ, A.F. 2014. Effect of moisture
content, temperature and exposure time on the physical stability of chitosan powder and tablets.
Drug development and industrial pharmacy, 40:730-742.
VIRIDÉN, A., ABRAHMSÉN-ALAMI, S., WITTGREN, B. & LARSSON, A. 2011. Release of
theophylline and carbamazepine from matrix tablets–consequences of HPMC chemical
heterogeneity. European journal of pharmaceutics and biopharmaceutics, 78:470-479.
VIRIDÉN, A., LARSSON, A., SCHAGERLÖF, H. & WITTGREN, B. 2010. Model drug release
from matrix tablets composed of HPMC with different substituent heterogeneity. International
journal of pharmaceutics, 401:60-67.
WAGSTAFF, A. 2002. Poverty and health sector inequalities. Bulletin of the world health
organization, 80:97-105.Diabetes fact sheet 2013.
http://www.who.int/diabetes/facts/world_figures/en Date of access: 3 March 2013
WILSON, I.D., NICHOLSON, J.K., CASTRO-PEREZ, J., GRANGER, J.H., JOHNSON, K.A., SMITH, B.W. & PLUMB, R.S. 2005. High resolution “ultra performance” liquid chromatography coupled to oa-TOF mass spectrometry as a tool for differential metabolic pathway profiling in functional genomic studies. Journal of proteome research, 4:591-599
WONG, A. C.-Y. 2002. Use of angle of repose and bulk densities for powder characterization
and the prediction of minimum fluidization and minimum bubbling velocities. Chemical
engineering science, 57:2635-2640.
YORK, P. 2013. Design of dosage forms. Aulton's pharmaceutics: The design and manufacture
of medicines, 7.
YOUNG, C.R., KOLENG, J.J. & MCGINITY, J.W. 2002. Production of spherical pellets by a hot-
melt extrusion and spheronization process. International journal of pharmaceutics, 242:87-92.
107
YANG, M., XIE, S., LI, Q., WANG, Y., CHANG, X., SHAN, L., SUN, L., HUANG, X. & GAO, C.,
2012. Effects of polyvinylpyrrolidone both as a binder and pore-former on the release of
sparingly water-soluble topiramate from ethylcellulose coated pellets. International journal of
pharmaceutics, 465:187-196
ZHANG, G.G., LAW, D., SCHMITT, E.A. & QIU, Y. 2004. Phase transformation considerations
during process development and manufacture of solid oral dosage forms. Advanced drug
delivery reviews, 56:371-390.
ZHANG, Y., LAW, Y. & CHAKRABARTI, S. 2003. Physical properties and compact analysis of
commonly used direct compression binders. aaps Pharmscitech, 4:489-499.
ZIMMET, P., ALBERTI, K. & SHAW, J. 2001. Global and societal implications of the diabetes
epidemic. Nature, 414:782-787.
108
ANNEXURE A
THERMOANALYSIS, MOISTURE CONTENT AND SIZE-DISTRIBUTION
109
MOISTURE CONTENT
Table A.I: Karl-Fischer titration values for moisture content of the donated Cassava starch
Donated Starch
Time
(min)
Moisture (%H2O) %Weight
Loss Run 1 Run 2 Average SD %RSD
Reference Sample
0 15.58 16.06 15.82 0.339 0.24 10.80
Moisture Content at 25°C
30 14.15 14.11 14.13 0.028 0.02 10.21
60 12.89 12.88 12.89 0.007 0.01 5.75
120 11.94 12.1 12.02 0.113 0.08 5.63
240 11.61 11.65 11.63 0.028 0.02 9.167
360 11.37 11.71 11.54 0.240 0.17 8.84
480 11.49 11.98 11.74 0.346 0.25 9.46
Moisture Content at 30°C
30 12.89 13.04 12.97 0.106 0.08 6.67
60 11.85 11.75 11.8 0.071 0.05 6.79
120 10.82 10.88 10.85 0.042 0.03 7.83
240 10.77 10.49 10.63 0.198 0.14 9.35
360 9.79 10.45 10.12 0.467 0.33 8.26
480 10.56 11.78 11.17 0.863 0.61 8.12
Moisture Content at 40°C
110
30 11.31 11.23 11.27 0.057 0.04 9.67
60 10.81 10.29 10.55 0.368 0.26 9.40
120 9.57 9.97 9.77 0.283 0.20 8.86
240 8.06 8.07 8.07 0.007 0.01 9.06
360 8.72 8.62 8.67 0.070 0.05 5.01
480 8.78 9.07 8.93 0.205 0.15 4.39
Moisture Content at 50°C
30 9.62 9.65 9.65 0.042 0.03 5.81
60 8.81 8.76 8.79 0.035 0.03 8.79
120 6.76 6.50 6.63 0.184 0.13 1.60
240 6.33 6.21 6.27 0.084 0.06 7.07
360 6.52 6.38 6.45 0.099 0.07 4.52
480 6.66 6.81 6.74 0.106 0.08 2.18
Table A.II: Karl-Fischer titration values for moisture content of the purchased Cassava starch
Purchased Starch
Time
(min)
Moisture (%H2O) %Weight
Loss Run 1 Run 2 Average SD %RSD
Reference Sample
0 11.95 11.73 11.84 0.156 0.11 7.89
Moisture Content at 25°C
30 12.28 11.81 12.05 0.332 0.24 4.47
60 12.61 12.51 12.56 0.071 0.05 10.19
111
120 12.1 12.37 12.24 0.191 0.14 7.44
240 12.59 12.42 12.51 0.120 0.09 7.87
360 12.39 11.62 12.01 0.544 0.39 10.64
480 11.64 11.82 11.73 0.127 0.09 6.61
Moisture Content at 30°C
30 11.63 11.75 11.69 0.085 0.06 10.10
60 11.58 11.39 11.49 0.134 0.10 9.94
120 11.2 10.63 10.92 0.403 0.29 8.79
240 10.14 9.97 10.06 0.120 0.09 3.43
360 10.19 10.18 10.19 0.007 0.01 8.79
480 10.69 10.74 10.72 0.035 0.03 3.88
Moisture Content at 40°C
30 10.13 9.82 9.98 0.219 0.11 4.49
60 9.46 9.25 9.36 0.148 0.07 3.91
120 8.26 8.29 8.28 0.021 0.01 8.47
240 9.06 8.51 8.79 0.389 0.19 10.06
360 8.02 8.01 8.02 0.007 0.00 3.86
480 8.36 8.26 8.31 0.070 0.04 8.86
Moisture Content at 50°C
30 8.91 8.87 8.89 0.028 0.02 4.97
60 7.72 7.71 7.72 0.007 0.01 8.19
120 7.21 7.04 7.13 0.120 0.09 3.65
240 6.29 6.81 6.55 0.368 0.26 2.92
112
360 6.06 6.68 6.37 0.438 0.31 7.15
480 6.84 7.13 6.99 0.205 0.15 4.25
MALVERN MASTERSIZER®
SIZE DISTRIBUTION
Particle Size Distribution
0.01 0.1 1 10 100 1000 10000
Particle Size (µm)
0
2
4
6
8
10
12
14
Volum
e (%
)
Cassava powder, 11 December 2014 11:36:16 AM
Particle Size Distribution
0.01 0.1 1 10 100 1000 10000
Particle Size (µm)
0
2
4
6
8
10
12
Volum
e (%
)
Cassava powder, 11 December 2014 11:53:39 AM
Particle Size Distribution
0.01 0.1 1 10 100 1000 10000
Particle Size (µm)
0
2
4
6
8
10
12
14
16
Volum
e (%
)
Formule 15, 11 December 2014 02:04:56 PM
Particle Size Distribution
0.01 0.1 1 10 100 1000 10000
Particle Size (µm)
0
2
4
6
8
10
12
14
16
18
Volum
e (%
)
Formule 11, 11 December 2014 01:39:54 PM
113
Particle Size Distribution
0.01 0.1 1 10 100 1000 10000
Particle Size (µm)
0
2
4
6
8
10
12
14
16 V
olum
e (%
)
Formule 21, 11 December 2014 02:08:14 PM
Particle Size Distribution
0.01 0.1 1 10 100 1000 10000
Particle Size (µm)
0
2
4
6
8
10
12
14
16
Volum
e (%
)
Formule 39, 11 December 2014 02:20:16 PM
Particle Size Distribution
0.01 0.1 1 10 100 1000 10000
Particle Size (µm)
0
2
4
6
8
10
12
14
16
Volum
e (%
)
Formule 46, 11 December 2014 02:35:02 PM
Particle Size Distribution
0.01 0.1 1 10 100 1000 10000
Particle Size (µm)
0
5
10
15
20
Volum
e (%
)
Formule 64, 11 December 2014 02:53:47 PM
Particle Size Distribution
0.01 0.1 1 10 100 1000 10000
Particle Size (µm)
0
5
10
15
20
Volum
e (%
)
Formule 74, 11 December 2014 03:04:24 PM
Figure A.I: Example graphs of size distribution graphs for starch powders and bead
formulations
Table A.III: Size distribution values starch powders and bead formulations
Donated starch Purchased starch C.G5.5.5 (Formula 15)
Run d(0.1) d(0.5) d(0.9) Run d(0.1) d(0.5) d(0.9) Run d(0.1) d(0.5) d(0.9)
1 7.57 14.09 24.97 1 7.52 15.68 27.56 1 11.92 809.98 1282.51
2 7.51 13.99 24.48 2 7.40 15.36 26.76 2 15.06 844.49 1292.73
3 7.53 14.02 24.56 3 7.27 15.10 26.19 3 22.25 800.28 1229.79
AVE 7.54 14.03 24.67 AVE 7.39 15.38 26.83 AVE 16.41 818.25 1268.35
SD 0.032 0.053 0.263 SD 0.125 0.293 0.688 SD 5.293 23.240 33.776
%RSD 0.43 0.38 1.07 %RSD 1.69 1.91 2.56 %RSD 32.26 2.84 2.66
M.G5.3.5 (Formula 11) M.G5.5.10 (Formula 21) M.G10.5.5 (Formula 39)
Run d(0.1) d(0.5) d(0.9) Run d(0.1) d(0.5) d(0.9) Run d(0.1) d(0.5) d(0.9)
1 130.48 915.14 1324.80 1 42.20 891.28 1353.65 1 625.00 974.89 1520.93
2 99.55 915.95 1326.34 2 33.04 909.48 1351.75 2 608.12 930.42 1303.36
3 80.79 939.39 1316.49 3 80.58 998.27 1461.19 3 583.63 955.66 1327.95
AVE 103.61 923.49 1322.54 AVE 51.94 933.01 1388.86 AVE 605.58 953.66 1384.08
SD 25.091 13.776 5.301 SD 25.223 57.246 62.647 SD 20.803 22.303 119.153
%RSD 24.22 1.49 0.40 %RSD 48.56 6.14 4.51 %RSD 3.44 2.34 8.61
M.G10.0.10 (Formula 46) M.G15.0.5 (Formula 64) M.G15.3.10 (Formula 74)
Run d(0.1) d(0.5) d(0.9) Run d(0.1) d(0.5) d(0.9) Run d(0.1) d(0.5) d(0.9)
1 685.42 1052.65 1579.03 1 635.24 875.93 1221.62 1 557.14 881.13 1236.32
2 665.75 1028.05 1559.55 2 635.91 888.56 1266.07 2 512.80 921.78 1295.57
3 649.22 1009.31 1540.53 3 673.76 920.55 1269.95 3 672.52 1013.07 1513.84
AVE 666.80 1030.00 1559.70 AVE 648.30 895.01 1252.55 AVE 580.82 938.66 1348.58
SD 18.123 21.7367 19.249 SD 22.048 22.998 26.857 SD 82.454 67.569 146.157
%RSD 2.72 2.11 1.23 %RSD 3.40 2.57 2.14 %RSD 14.20 7.20 10.84
116
ANNEXURE B
FLOW AND PHYSICAL PROPERTIES
FLOW PROPERTIES
Table B.I: Time and flow rate for Cassava starch powders and beads
Samples Mass
(g)
Time (s) Flow Rate (g.s-1)
1 2 3 Ave SD 1 2 3 Ave SD
Donated 100.14 No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
Purchased 101.07 No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
M.G5.3.5 99.59 22.00 20.00 21.00 21.00 1.000 4.53 4.98 4.74 4.75 0.226
M.G5.5.10 100.84 22.00 21.00 21.00 21.33 0.577 4.58 4.80 4.80 4.73 0.126
M.G10.5.5 99.72 22.00 21.00 22.00 21.67 0.577 4.53 4.75 4.53 4.60 0.125
M.G10.0.10 99.97 19.00 19.00 20.00 19.33 0.577 5.26 5.26 5.00 5.17 0.152
M.G15.0.5 100.02 26.00 26.00 27.00 26.33 0.577 3.85 3.85 3.70 3.80 0.082
M.G15.3.10 100.73 28.00 28.00 27.00 27.67 0.577 3.60 3.60 3.73 3.64 0.077
C.G5.5.5 100.10 20.00 20.00 21.00 20.33 0.577 5.01 5.01 4.77 4.93 0.138
Table B.II: Parameters relating to angle of repose, angle of repose and critical orifice diameter
Samples Mass
(g)
h (mm) r (mm)
1 2 3 Ave SD 1 2 3 Ave SD
Donated 100.14 No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
Purchased 101.07 No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
M.G5.3.5 99.59 3.00 3.10 2.90 3.00 0.100 5.95 6.15 5.85 5.98 0.153
M.G5.5.10 100.84 3.00 3.00 3.10 3.03 0.058 5.00 6.21 5.80 5.67 0.615
M.G10.5.5 99.72 3.10 2.70 2.90 2.90 0.200 6.05 6.00 6.00 6.02 0.029
M.G10.0.10 99.97 3.20 3.30 3.30 3.27 0.058 6.20 6.25 5.80 6.08 0.247
M.G15.0.5 100.02 3.10 3.50 3.30 3.30 0.200 5.95 5.60 5.75 5.77 0.176
M.G15.3.10 100.73 3.40 3.40 3.50 3.43 0.058 5.80 5.90 6.05 5.92 0.126
C.G5.5.5 100.10 2.90 3.00 3.00 2.97 0.058 6.00 5.50 6.20 6.02 0.361
Samples Mass
(g)
AoR (°) COD (mm)
1 2 3 Ave SD 1 2 3 Ave SD
Donated 100.14 No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
16.00 16.00 16.00 16.00 0.000
Purchased 101.07 No
Flow
No
Flow
No
Flow
No
Flow
No
Flow
16.00 16.00 16.00 16.00 0.000
M.G5.3.5 99.59 26.76 26.75 26.37 26.63 0.223 5.00 6.00 6.00 5.67 0.577
M.G5.5.10 100.84 30.96 25.78 28.12 28.29 2.594 6.00 6.00 6.00 6.00 0.000
M.G10.5.5 99.72 27.13 24.23 25.80 25.72 1.453 6.00 6.00 6.00 6.00 0.000
M.G10.0.10 99.97 27.30 27.83 29.64 28.26 1.226 6.00 7.00 7.00 6.67 0.577
M.G15.0.5 100.02 27.52 32.01 29.85 29.79 2.243 6.00 6.00 6.00 6.00 0.000
M.G15.3.10 100.73 30.38 29.95 30.05 30.13 0.223 6.00 6.00 6.00 6.00 0.000
C.G5.5.5 100.10 25.61 26.57 26.57 26.25 0.551 6.00 6.00 6.00 6.00 0.000
DENSITIES & COMPRESSIBILITY
Table B.III Volumes, densities and compressibility data for both starches and bead formulations
Bulk
Samples Mass
(g)
Volume (cm3) Density (g.cm-3)
1 2 3 Ave SD 1 2 3 Ave SD
Donated 100.14 168.00 166.00 164.00 166.00 2.000 0.60 0.60 0.61 0.60 0.007
Purchased 101.07 192.00 182.00 180.00 184.67 6.429 0.53 0.56 0.56 0.55 0.019
M.G5.3.5 99.59 138.00 124.00 122.00 128.00 8.718 0.72 0.80 0.82 0.78 0.051
M.G5.5.10 100.84 120.00 122.00 122.00 121.33 1.155 0.84 0.83 0.83 0.83 0.008
M.G10.5.5 99.72 126.00 126.00 124.00 125.33 1.155 0.79 0.79 0.80 0.80 0.007
M.G10.0.10 99.97 124.00 122.00 125.00 123.67 1.528 0.81 0.82 0.80 0.81 0.010
M.G15.0.5 100.02 120.00 121.00 121.00 120.67 0.577 0.83 0.83 0.83 0.83 0.004
M.G15.3.10 100.73 128.00 127.00 125.00 126.67 1.528 0.79 0.79 0.81 0.80 0.010
C.G5.5.5 100.10 121.00 122.00 123.00 122.00 1.000 0.83 0.82 0.81 0.82 0.007
Tapped
Samples Mass
(g)
Volume (cm3) Density (g.cm-3)
1 2 3 Ave SD 1 2 3 Ave SD
Donated 100.14 124.00 122.00 118.00 121.33 3.055 0.81 0.82 0.85 0.83 0.021
Purchased 101.07 130.00 124.00 120.00 124.67 5.033 0.78 0.82 0.84 0.81 0.033
M.G5.3.5 99.59 110.00 110.00 112.00 110.67 1.155 0.91 0.91 0.89 0.90 0.009
M.G5.5.10 100.84 116.00 114.00 114.00 114.67 1.155 0.87 0.88 0.88 0.88 0.009
M.G10.5.5 99.72 114.00 114.00 114.00 114.00 0.000 0.87 0.87 0.87 0.87 0.000
M.G10.0.10 99.97 111.00 110.00 111.00 110.67 0.577 0.90 0.91 0.90 0.90 0.005
M.G15.0.5 100.02 115.00 115.00 116.00 115.33 0.577 0.87 0.87 0.86 0.87 0.004
M.G15.3.10 100.73 116.00 116.00 116.00 116.00 0.000 0.87 0.87 0.87 0.87 0.000
C.G5.5.5 100.10 114.00 114.00 115.00 114.33 0.577 0.88 0.88 0.87 0.88 0.004
Packed
Samples Mass
(g)
Volume (cm3) Packed Density (g.cm-3)
1 2 3 Ave SD 1 2 3 Ave SD
Donated 100.14 44.00 44.00 46.00 44.67 1.155 2.28 2.28 2.18 2.24 0.057
Purchased 101.07 62.00 58.00 60.00 60.00 2.000 1.63 1.74 1.68 1.69 0.056
M.G5.3.5 99.59 28.00 14.00 10.00 17.33 9.452 3.56 7.11 9.96 6.88 3.208
M.G5.5.10 100.84 4.00 8.00 8.00 6.67 2.309 25.21 12.61 12.61 16.81 7.278
M.G10.5.5 99.72 12.00 12.00 10.00 11.33 1.155 8.31 8.31 9.97 8.86 0.960
M.G10.0.10 99.97 13.00 12.00 14.00 13.00 1.000 7.69 8.33 7.14 7.72 0.596
M.G15.0.5 100.02 5.00 6.00 5.00 5.33 0.577 20.00 16.67 20.00 18.89 1.925
M.G15.3.10 100.73 12.00 11.00 9.00 10.67 1.528 8.39 9.16 11.19 9.58 1.446
C.G5.5.5 100.10 7.00 8.00 8.00 7.67 0.577 14.30 12.51 12.51 13.11 1.032
Compressibility
Samples Mass
(g)
Carr's index Hausner Ration
1 2 3 Ave SD 1 2 3 Ave SD
Donated 100.14 0.26 0.27 0.28 0.27 0.010 1.35 1.36 1.39 1.37 0.019
Purchased 101.07 0.32 0.32 0.33 0.32 0.008 1.48 1.47 1.50 1.48 0.017
M.G5.3.5 99.59 0.20 0.11 0.08 0.13 0.063 1.25 1.13 1.09 1.16 0.087
M.G5.5.10 100.84 0.06 0.07 0.07 0.06 0.004 1.06 1.07 1.07 1.07 0.005
M.G10.5.5 99.72 0.03 0.07 0.07 0.05 0.019 1.03 1.07 1.07 1.06 0.021
M.G10.0.10 99.97 0.10 0.10 0.08 0.09 0.008 1.11 1.11 1.09 1.10 0.010
M.G15.0.5 100.02 0.10 0.10 0.11 0.11 0.007 1.12 1.11 1.13 1.12 0.009
M.G15.3.10 100.73 0.04 0.05 0.04 0.04 0.005 1.04 1.05 1.04 1.05 0.005
C.G5.5.5 100.10 0.09 0.09 0.07 0.08 0.011 1.10 1.09 1.08 1.09 0.013
121
SWELLING AND EROSION
Table B.IV: Swelling and erosion data for Avicel® bead formulations
Time
(min) Parameter
M.G5.3.5
Run 1 Run 2 Run 3 Average SD
Initial mass 251.50 249.00 249.50 250.00 0.500
30
Mass 1429.76 1424.00 1416.76 1423.51 4.045
%Swelling 102.71 102.69 102.68 102.69 0.009
%Erosion 177.37 175.20 173.94 175.50 0.829
60
Mass 1230.20 1205.50 1198.80 1211.50 6.353
%Swelling 102.25 102.19 102.18 102.20 0.015
%Erosion 176.58 174.34 173.09 174.67 0.834
90
Mass 1269.80 1218.80 1214.30 1234.30 10.492
%Swelling 102.34 102.22 102.21 102.26 0.024
%Erosion 176.74 174.39 173.15 174.76 0.844
120
Mass 1259.60 1181.40 1178.70 1206.57 15.369
%Swelling 102.31 102.14 102.13 102.19 0.035
%Erosion 176.70 174.25 173.01 174.65 0.854
180
Mass 1923.80 1873.10 1857.90 1884.93 13.552
%Swelling 103.84 103.72 103.69 103.75 0.031
%Erosion 179.33 176.96 175.66 177.32 0.874
240
Mass 1929.70 1920.90 1910.30 1920.30 5.954
%Swelling 103.85 103.83 103.81 103.83 0.014
%Erosion 179.36 177.15 175.86 177.45 0.846
360
Mass 1832.40 1839.80 1819.30 1830.50 10.265
%Swelling 103.63 103.65 103.60 103.63 0.024
%Erosion 178.97 176.83 175.51 177.10 0.853
480
Mass 1839.20 1766.80 1739.20 1781.73 21.579
%Swelling 103.65 103.48 103.42 103.51 0.050
%Erosion 179.00 176.54 175.19 176.91 0.904
600 Mass 2010.10 1926.70 1905.70 1947.50 20.900
122
%Swelling 104.04 103.85 103.80 103.89 0.048
%Erosion 179.67 177.17 175.84 177.56 0.901
720
Mass 1883.60 1812.20 1801.70 1832.50 15.658
%Swelling 103.75 103.58 103.56 103.63 0.036
%Erosion 179.17 176.72 175.44 177.12 0.875
Dried Mass 172.70 170.60 169.40 170.90 0.794
Time (min)
Parameter M.G5.5.10
Run 1 Run 2 Run 3 Average SD
Initial mass 250.30 249.70 251.20 250.40 0.751
30
Mass 1367.50 1301.80 1284.30 1317.87 16.788
%Swelling 102.58 102.43 102.39 102.47 0.039
%Erosion 175.63 173.93 175.60 175.05 0.852
60
Mass 1421.70 1404.10 1348.80 1391.53 28.989
%Swelling 102.71 102.67 102.54 102.64 0.067
%Erosion 175.84 174.33 175.86 175.34 0.776
90
Mass 1446.30 1375.30 1353.30 1391.63 19.236
%Swelling 102.77 102.60 102.55 102.64 0.045
%Erosion 175.94 174.22 175.88 175.34 0.845
120
Mass 1621.20 1564.30 1515.60 1567.03 28.938
%Swelling 103.17 103.04 102.93 103.05 0.067
%Erosion 176.63 174.96 176.52 176.04 0.797
180
Mass 1711.20 1663.00 1628.30 1667.50 21.451
%Swelling 103.38 103.27 103.19 103.28 0.050
%Erosion 176.99 175.35 176.97 176.43 0.824
240
Mass 1541.40 1503.40 1495.10 1513.30 9.112
%Swelling 102.99 102.90 102.88 102.92 0.021
%Erosion 176.31 174.72 176.44 175.83 0.869
360
Mass 1465.70 1469.00 1460.40 1465.03 4.304
%Swelling 102.81 102.82 102.80 102.81 0.010
%Erosion 176.01 174.59 176.30 175.63 0.863
480
Mass 1642.20 1573.00 1500.20 1571.80 41.689
%Swelling 103.22 103.06 102.89 103.06 0.096
%Erosion 176.71 175.00 176.46 176.06 0.755
600
Mass 1774.60 1741.00 1743.70 1753.10 6.352
%Swelling 103.53 103.45 103.46 103.48 0.014
%Erosion 177.24 175.66 177.42 176.77 0.894
720 Mass 1562.00 1525.30 1501.30 1529.53 15.226
%Swelling 103.04 102.95 102.89 102.96 0.033
123
%Erosion 176.40 174.81 176.46 175.89 0.840
Dried Mass 171.20 169.80 171.50 170.83 0.857
Time (min)
Parameter M.G10.5.5
Run 1 Run 2 Run 3 Average SD
Initial mass 250.40 251.10 250.30 250.60 0.404
30
Mass 1257.10 1251.00 1222.60 1243.57 14.728
%Swelling 102.29 102.27 102.21 102.26 0.033
%Erosion 174.30 178.06 175.80 176.05 1.237
60
Mass 1051.80 1039.50 1019.60 1036.97 10.832
%Swelling 101.82 101.79 101.75 101.79 0.025
%Erosion 173.50 177.22 175.01 175.24 1.215
90
Mass 1247.40 1227.40 1223.80 1232.87 4.565
%Swelling 102.27 102.22 102.21 102.23 0.010
%Erosion 174.26 177.96 175.81 176.01 1.191
120
Mass 1234.10 1213.10 1214.40 1220.53 3.970
%Swelling 102.24 102.19 102.19 102.21 0.009
%Erosion 174.21 177.91 175.77 175.96 1.183
180
Mass 724.70 675.90 654.40 685.00 15.713
%Swelling 101.08 100.97 100.92 100.99 0.036
%Erosion 172.24 175.78 173.58 173.87 1.197
240
Mass 664.70 688.00 667.70 673.47 10.461
%Swelling 100.94 100.99 100.95 100.96 0.024
%Erosion 172.00 175.83 173.63 173.82 1.218
360
Mass 341.40 352.90 354.70 349.67 2.550
%Swelling 100.21 100.23 100.24 100.23 0.006
%Erosion 170.75 174.50 172.41 172.56 1.170
480
Mass 637.10 588.60 571.60 599.10 13.877
%Swelling 100.88 100.77 100.73 100.79 0.032
%Erosion 171.90 175.44 173.26 173.53 1.188
600
Mass 765.70 737.30 714.50 739.17 13.734
%Swelling 101.17 101.11 101.05 101.11 0.031
%Erosion 172.40 176.03 173.82 174.08 1.207
720
Mass 555.10 552.50 542.90 550.17 5.007
%Swelling 100.69 100.69 100.66 100.68 0.011
%Erosion 171.58 175.29 173.14 173.34 1.189
Dried Mass 170.40 174.10 172.00 172.17 1.167
Time (min)
Parameter M.G10.0.10
Run 1 Run 2 Run 3 Average SD
Initial mass 250.10 251.70 251.20 251.00 0.361
30 Mass 1303.40 1212.70 1202.60 1239.57 19.107
124
%Swelling 102.49 102.28 102.25 102.34 0.045
%Erosion 174.34 177.04 174.54 175.31 1.279
60
Mass 1296.30 1273.50 1216.80 1262.20 30.010
%Swelling 102.47 102.42 102.29 102.39 0.071
%Erosion 174.31 177.29 174.60 175.40 1.380
90
Mass 1409.20 1365.10 1341.40 1371.90 16.011
%Swelling 102.74 102.64 102.58 102.65 0.038
%Erosion 174.76 177.66 175.10 175.84 1.317
120
Mass 1459.10 1402.80 1417.80 1426.57 12.019
%Swelling 102.86 102.73 102.76 102.78 0.028
%Erosion 174.96 177.82 175.41 176.06 1.244
180
Mass 898.50 865.50 874.70 879.57 7.144
%Swelling 101.53 101.46 101.48 101.49 0.017
%Erosion 172.71 175.62 173.22 173.85 1.243
240
Mass 1008.60 980.60 983.90 991.03 5.333
%Swelling 101.79 101.73 101.74 101.75 0.013
%Erosion 173.15 176.09 173.66 174.30 1.258
360
Mass 1398.70 1460.40 1457.60 1438.90 11.689
%Swelling 102.72 102.86 102.86 102.81 0.028
%Erosion 174.72 178.05 175.57 176.11 1.304
480
Mass 891.90 882.80 865.90 880.20 9.100
%Swelling 101.52 101.50 101.46 101.49 0.022
%Erosion 172.68 175.69 173.18 173.85 1.297
600
Mass 961.80 916.60 820.10 899.50 51.493
%Swelling 101.68 101.58 101.35 101.54 0.122
%Erosion 172.96 175.83 173.00 173.93 1.441
720
Mass 1168.20 1151.60 1121.50 1147.10 16.236
%Swelling 102.17 102.13 102.06 102.12 0.038
%Erosion 173.79 176.79 174.22 174.93 1.328
Dried Mass 170.10 173.10 170.70 171.30 1.249
Time (min)
Parameter M.G15.0.5
Run 1 Run 2 Run 3 Average SD
Initial mass 250.40 251.20 251.10 250.90 0.153
30
Mass 1164.10 1143.20 1085.10 1130.80 30.599
%Swelling 102.14 102.10 101.96 102.07 0.072
%Erosion 175.28 169.48 175.27 173.34 2.950
60
Mass 1166.20 1114.90 1083.90 1121.67 20.137
%Swelling 102.15 102.03 101.96 102.04 0.047
%Erosion 175.29 169.37 175.26 173.31 3.004
90 Mass 1069.50 1042.90 1038.20 1050.20 6.047
125
%Swelling 101.92 101.86 101.85 101.88 0.014
%Erosion 174.90 169.09 175.08 173.02 3.046
120
Mass 1137.90 1180.40 1151.60 1156.63 15.382
%Swelling 102.08 102.18 102.11 102.13 0.036
%Erosion 175.17 169.62 175.54 173.44 3.000
180
Mass 1447.80 1411.90 1382.40 1414.03 17.680
%Swelling 102.81 102.73 102.66 102.73 0.041
%Erosion 176.42 170.52 176.47 174.47 3.025
240
Mass 1259.60 1257.00 1242.00 1252.87 7.748
%Swelling 102.37 102.36 102.33 102.35 0.018
%Erosion 175.66 169.92 175.90 173.83 3.038
360
Mass 1368.80 1359.70 1341.70 1356.73 9.651
%Swelling 102.62 102.60 102.56 102.60 0.023
%Erosion 176.10 170.32 176.30 174.24 3.040
480
Mass 1426.10 1354.60 1325.60 1368.77 22.004
%Swelling 102.76 102.59 102.52 102.62 0.052
%Erosion 176.33 170.30 176.24 174.29 3.028
600
Mass 1502.50 1466.20 1433.50 1467.40 19.235
%Swelling 102.94 102.85 102.78 102.86 0.045
%Erosion 176.64 170.73 176.67 174.68 3.023
720
Mass 1290.60 1265.90 1263.20 1273.23 5.192
%Swelling 102.44 102.38 102.38 102.40 0.012
%Erosion 175.79 169.95 175.99 173.91 3.065
Dried Mass 171.60 166.00 171.90 169.83 2.994
Time (min)
Parameter M.G15.0.5
Run 1 Run 2 Run 3 Average SD
Initial mass 250.20 250.90 251.30 250.80 0.265
30
Mass 1323.30 1290.90 1304.30 1306.17 8.328
%Swelling 102.51 102.44 102.47 102.47 0.020
%Erosion 175.50 176.40 175.63 175.84 0.395
60
Mass 1112.00 1126.50 1090.60 1109.70 17.962
%Swelling 102.02 102.05 101.97 102.01 0.042
%Erosion 174.65 175.73 174.77 175.05 0.494
90
Mass 1065.40 1062.20 1073.80 1067.13 5.822
%Swelling 101.91 101.90 101.93 101.91 0.014
%Erosion 174.47 175.47 174.70 174.88 0.402
120
Mass 1216.90 1171.00 1174.10 1187.33 8.675
%Swelling 102.26 102.16 102.16 102.19 0.020
%Erosion 175.08 175.91 175.11 175.37 0.411
180 Mass 1377.60 1367.60 1359.60 1368.27 4.823
126
%Swelling 102.64 102.62 102.60 102.62 0.011
%Erosion 175.72 176.71 175.85 176.09 0.440
240
Mass 1217.80 1229.00 1198.10 1214.97 15.472
%Swelling 102.27 102.29 102.22 102.26 0.036
%Erosion 175.08 176.15 175.20 175.48 0.485
360
Mass 1124.10 1109.00 1105.40 1112.83 3.717
%Swelling 102.05 102.01 102.00 102.02 0.009
%Erosion 174.70 175.66 174.83 175.07 0.428
480
Mass 1340.30 1294.60 1283.50 1306.13 11.317
%Swelling 102.55 102.45 102.42 102.47 0.027
%Erosion 175.57 176.41 175.55 175.84 0.439
600
Mass 1416.40 1356.70 1321.80 1364.97 22.912
%Swelling 102.73 102.59 102.51 102.61 0.054
%Erosion 175.88 176.66 175.70 176.08 0.484
720
Mass 1159.10 1189.70 1174.10 1174.30 8.949
%Swelling 102.13 102.20 102.16 102.16 0.021
%Erosion 174.84 175.99 175.11 175.31 0.460
Dried Mass 171.20 172.20 171.40 171.60 0.416
Table B.V: Friability parameters and data
Sample Parameters 1 2 3 Ave SD
M.G5.3.5 W0 3.00 3.00 3.01 3.00 0.005
W1 2.84 3.01 3.01 2.96 0.100
%Friability 5.33 0.00 0.00 1.78 3.079
M.G5.5.10 W0 3.00 3.00 3.00 3.00 0.001
W1 3.00 2.99 3.01 3.00 0.008
%Friability 0.00 0.37 0.00 0.12 0.212
M.G10.5.5 W0 3.00 3.00 3.00 3.00 0.001
W1 3.01 3.01 3.01 3.01 0.003
%Friability 0.00 0.00 0.00 0.00 0.000
M.G10.0.10 W0 3.00 3.00 3.00 3.00 0.001
W1 2.98 2.99 2.98 2.98 0.005
%Friability 0.63 0.47 0.87 0.66 0.201
M.G15.0.5 W0 3.00 3.00 3.01 3.00 0.002
W1 3.01 3.01 3.02 3.01 0.005
%Friability 0.00 0.00 0.00 0.00 0.000
M.G15.3.10 W0 3.00 3.00 3.00 3.00 0.002
W1 3.01 3.04 3.01 3.02 0.015
%Friability 0.00 0.00 0.00 0.00 0.000
127
C.G.5.5.5 W0 3.00 3.00 3.00 3.00 0.001
W1 2.91 2.97 2.95 2.94 0.027
%Friability 2.87 1.10 1.90 1.96 0.885
Table B.VI Disintegration times
Samples
Disintegration time (s)
Ave SD Vessel 1
Vessel 2
Vessel 3
Vessel 4
Vessel 5
Vessel 6
M.G5.3.5 241.80 247.80 259.20 240.60 252.00 249.00 248.40 6.852
M.G5.5.10 300.00 258.00 313.80 420.60 378.00 480.00 358.40 83.090
M.G10.5.5 420.00 510.00 486.00 498.00 540.00 486.00 490.00 39.739
M.G10.0.10 275.40 336.00 288.00 258.00 330.00 354.00 306.90 38.313
M.G15.0.5 600.00 606.00 564.00 570.00 330.00 384.00 509.00 120.085
M.G15.3.10 300.60 270.00 510.00 396.00 450.00 330.00 376.10 92.595
C.G5.5.5 210.00 270.00 330.00 300.00 294.00 198.00 267.00 52.547
128
ANNEXURE C
DISSOLUTION STUDIES
129
LINEARITY AND VALIDATION
Table C.I: Linearity and validation data for gliclazide in acidic medium
Medium Acidic Day 1 Run 1 Intraday
Standard Gliclazide (µg.ml-1)
Average Absorbance
SD %RSD Mass (mg)
25.1
1 2.01 0.06 0.00 0.08
Regression 2 5.02 0.19 0.00 0.05
3 10.04 0.39 0.00 0.04
4 20.08 0.74 0.00 0.03 m 0.04
5 30.12 1.13 0.06 4.01 c -0.00
6 40.16 1.49 0.0 0.06 r 0.9998
Medium Acidic Day 1 Run 2 Intraday
Standard Gliclazide (µg.ml-1)
Average Absorbance
SD %RSD Mass (mg)
25.1
1 2 0.54 0.00 0.04
Regression 2 5 0.095 0.00 0.04
3 10 0.46 0.00 0.02
4 20 0.83 0.00 0.00 m 0.19
5 30 1.20 0.00 0.01 c -0.10
6 40 1.59 0.00 0.10 r 0.9999
Medium Acidic Day 1 Run 3 Intraday
Standard Gliclazide (µg.ml-1)
Average Absorbance
SD %RSD Mass (mg)
25.1
1 2.01 0.06 0.01 0.10
Regression 2 5.02 0.10 0.00 0.00
3 10.04 0.43 0.12 0.20
4 20.08 0.81 0.46 0.00 m 0.18
5 30.12 1.23 0.00 2.00 c -0.10
6 40.16 1.50 1.29 1.07 r 0.9997
Medium Acidic Day 2 Run 1 Interday
Standard Gliclazide (µg.ml-1)
Average Absorbance
SD %RSD Mass (mg)
25.1
1 2.01 0.08 0.01 12.63
Regression 2 5.02 0.21 0.00 0.19
3 10.04 0.43 0.01 1.06
4 20.08 0.82 0.03 2.41 m 0.04
5 30.12 1.20 0.21 12.40 c 0.02
6 40.16 1.59 0.00 0.21 r 0.9997
Medium Acidic Day 3 Run 1 Interday
130
Standard Gliclazide (µg.ml-1)
Average Absorbance
SD %RSD Mass (mg)
25.1
1 2.01 -0.04 0.00 -0.19
Regression 2 5.02 0.08 0.00 0.09
3 10.04 0.30 0.00 0.22
4 20.08 0.67 0.00 0.26 m 0.04
5 30.12 1.06 0.00 0.01 c -0.11
6 40.16 1.46 0.00 0.11 r 0.9999
Table C.II Linearity and validation data for gliclazide in alkaline medium
Medium Alkaline Day 1 Run 1 Intraday
Standard Gliclazide (µg.ml-1)
Average Absorbance
SD %RSD Mass (mg)
25
1 2 0.00 0.00 7.19
Regression 2 5 0.15 0.00 0.33
3 10 0.32 0.01 4.29
4 20 0.70 0.00 0.22 m 0.03
5 30 1.07 0.00 0.04 c -0.06
6 40 1.49 0.00 0.05 r 0.9995
Medium Alkaline Day 1 Run 2 Intraday
Standard Gliclazide (µg.ml-1)
Average Absorbance
SD %RSD Mass (mg)
25
1 2 0.01 0.00 0.01
Regression 2 5 0.23 0.00 0.09
3 10 0.33 0.00 2.70
4 20 0.59 0.00 0.00 m 0.19
5 30 1.10 0.02 0.01 c -0.30
6 40 1.53 0.01 0.00 r 0.992
Medium Alkaline Day 1 Run 3 Intraday
Standard Gliclazide (µg.ml-1)
Average Absorbance
SD %RSD Mass (mg)
25.1
1 2.01 0.18 0.00 0.03
Regression 2 5.02 0.24 0.00 0.30
3 10.04 0.33 0.00 0.87
4 20.08 0.77 1,00 0.99 m 0.1532
5 30,12 1.01 0.20 0.01 c -0.2698
6 40.16 1.40 0.00 0.00 r 0.9997
Medium Alkaline Day 2 Run 1 Interday
Standard Gliclazide (µg.ml-1)
Average Absorbance
SD %RSD Mass (mg)
24.9
131
1 1.992 0.17 0.00 0.33
Regression 2 4.98 0.27 0.00 0.04
3 9.96 0.43 0.00 0.05
4 19.92 0.77 0.00 0.05 m 0.03
5 29.88 1.08 0.01 0.49 c 0.10
6 39.84 1.46 0.00 0.15 r 0.9996
Medium Alkaline Day 3 Run 1 Interday
Standard Gliclazide (µg.ml-1)
Average Absorbance
SD %RSD Mass (mg)
25
1 2 0.00 0.00 3.30
Regression 2 5 0.11 0.00 0.33
3 10 0.33 0.00 0.04
4 20 0.69 0.00 0.01 m 0.04
5 30 1.07 0.01 0.50 c -0.06
6 40 1.42 0.15 7.30 r 0.9997
132
DISSOLUTION DATA
Table C.III: Dissolution data for bead formulations and Diamicron®
Time (min)
M.G5.3.5 M.G5.5.10 M.G10.5.5 M.G10.0.10
Ave SD %RSD Ave SD %RSD Ave SD %RSD Ave SD
0 0.00 0.000 0 0.00 0.000 0.00 0 0 0 0 0
2.5 3.55 1.314 36.99 3.07 0.263 8.58 19.97 1.074 5.38 5.93 0.222
5.0 6.11 0.933 15.28 4.80 0.771 16.06 21.60 0.659 3.05 7.54 0.948
7.5 8.86 0.889 10.03 7.22 1.639 22.71 23.44 0.825 3.52 9.86 0.766
15 11.8
8 2.333 19.63 13.62 2.828 20.76 26.80 3.898 14.54 11.07 0.897
30 16.8
4 2.983 17.71 26.71 9.634 36.07 31.70 3.947 12.45 16.56 1.104
60 20.3
8 4.393 21.56 42.06 2.831 6.73 37.33 1.876 5.03 19.93 1.438
90 26.5
3 3.096 11.67 48.04 2.457 5.11 45.88 0.979 2.13 26.48 1.964
120 30.5
8 1.891 6.18 55.03 5.574 10.13 51.90 3.122 6.02 34.22 0.619
180 32.1
9 2.223 6.91 59.66 3.082 5.17 54.96 3.104 5.65 39.62 3.415
240 39.7
3 2.516 6.33 63.26 3.535 5.59 64.90 4.356 6.71 44.16 1.413
360 44.4
1 2.899 6.53 68.63 2.061 3.00 66.53 2.965 4.46 69.14 5.569
480 59.0
4 10.281 17.41 84.77 9.409 11.10 76.50
11.73
0 15.33 81.41 1.999
600 87.0
2 7.824 8.99 94.29 2.784 2.95 90.93 6.453 7.10 87.17 0.586
720 92.8
1 4.433 4.78 97.55 2.058 2.11 98.86 0.424 0.43 95.96 1.517
735 100.00
0.000 0.00 100.0
0 0.000 0.00 100.00 0.000 0.00 100.00 0.000
Time (min)
M.G15.0.5 M.G15.3.10 C.G5.5.5 Diamicron®
Ave SD %RSD Ave SD %RSD Ave SD %RSD Ave SD
0 0 0 0 0 0 0 0 0 0 0 0
2.5 11.33 2.061 18.19 2.92 0.379 12.97 32.63 9.765 29.93 11.92 4.847
133
5.0 13.09 2.118 16.18 4.89 1.356 27.73 39.57 7.155 18.08 12.54 1.311
7.5 15.27 1.481 9.70 6.45 0.622 9.65 44.57 7.746 17.38 19.43 4.618
15 16.10 1.376 8.55 8.95 2.372 26.49 60.44 11.27
7 18.66 22.62 2.768
30 18.89 1.002 5.30 14.65 4.239 28.94 63.88 7.676 12.02 25.29 1.679
60 23.81 1.320 5.54 22.17 1.648 7.43 66.62 8.967 13.46 28.35 2.376
90 27.86 0.806 2.89 31.67 0.602 1.90 69.37 7.784 11.22 36.91 7.244
120 32.51 2.732 8.40 35.95 4.587 12.76 70.87 8.215 11.59 37.62 1.353
180 34.66 4.384 12.65 43.13 3.708 8.60 74.67 8.698 11.65 45.51 4.353
240 42.60 3.490 8.19 65.77 3.910 5.94 78.94 9.059 11.48 74.64 0.857
360 56.30 5.872 10.43 73.23 6.101 8.33 81.85 8.301 10.14 77.18 2.306
480 67.27 4.423 6.57 82.71 3.994 4.83 88.55 8.407 9.49 83.48 4.250
600 86.71 8.099 9.34 84.54 7.178 8.49 93.03 3.613 3.88 88.70 6.583
720 92.46 5.926 6.41 92.70 5.834 6.29 94.64 3.849 4.07 95.07 1.538
735 100.0
0 0.000 0.00
100.0
0 0.000 0.00 100.00 0.000 0.00 100.00 0.000